Marine Natural Products with Activities against Prostate Cancer: Recent Discoveries.

Prostate cancer is the most common cancer in men, with over 52,000 new cases diagnosed every year. Diagnostics and early treatment are potentially hindered by variations in screening protocols, still largely reliant on serum levels of acid phosphatase and prostate-specific antigen, with tumour diagnosis and grading relying on histopathological examination. Current treatment interventions vary in terms of efficacy, cost and severity of side effects, and relapse can be aggressive and resistant to the current standard of care. For these reasons, the scientific community is looking for new chemotherapeutic agents. This review reports compounds and extracts derived from marine organisms as a potential source of new drugs against prostate cancer. Whilst there are several marine-derived compounds against other cancers, such as multiple myeloma, leukemia, breast and lung cancer, already available in the market, the presently collated findings show how the marine environment can be considered to hold potential as a new drug source for prostate cancer, as well. This review presents information on compounds presently in clinical trials, as well as new compounds/extracts that may enter trials in the future. We summarise information regarding mechanisms of action and active concentrations.

The Long Non-Coding RNA H19 Drives the Proliferation of Diffuse Intrinsic Pontine Glioma with H3K27 Mutation.

Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.

Galactose:PEGamine coated gold nanoparticles adhere to filopodia and cause extrinsic apoptosis.

Ultra-small gold nanoparticles, surface functionalised with a 50 : 50 ratio of a thiolated α-galactose derivative and a thiolated hexaethylene glycol amine, are toxic to HSC-3 oral squamous carcinoma cells. Differences in nanoparticle toxicity were found to be related to the synthesis duration, with 1 h reaction nanoparticles being less toxic than 5 h reaction nanoparticles. The ligand density decreased with longer reaction time, although the size, charge and ligand ratio remained similar. The concentration of sodium borohydride in the reaction decreased logarithmically over 5 h but remained within a concentration range sufficient to desorb weakly bound ligands, possibly explaining the observed gradual decrease in ligand density. Nanoparticle toxicity was abrogated by inhibition of either caspase 3/7 or caspase 8, but not by inhibition of caspase 9, consistent with extrinsic apoptosis. Electron microscopic analysis of cellular uptake demonstrated predominantly cytoplasmic localization. However, when energy-dependent transport was inhibited, by lowering the temperature to 4 °C, a remarkable adhesion of nanoparticles to filopodia was observed. Inhibition of filopodial assembly with a fascin inhibitor prevented nanoparticle adhesion to HSC-3 cells at 4 °C, while fascin inhibition at 37 °C resulted in less cytoplasmic uptake. More adhesion to HSC-3 filopodia was seen with the higher toxicity 5 h reaction time nanoparticles than with the 1 h nanoparticles. By including two further cell types (HaCaT keratinocytes and hCMEC/D3 endothelial cells), a pattern of increasing toxicity with filopodial binding of 5 h reaction nanoparticles became apparent.

Photodynamic therapy and diagnosis: Principles and comparative aspects.

Photodynamic therapy (PDT) is an evolving method of treating superficial tumours that is non-invasive and carries minimal risk of toxicity. It combines tumour-selective photosensitiser dyes, tissue oxygen and targeted illumination to generate cytotoxic reactive oxygen species (ROS) within the tumour. In addition to directly acting on tumour cells, PDT damages and restricts tumour microvasculature, and causes a local inflammatory response that stimulates an immune response against the tumour. Unlike surgery or radiotherapy, the surrounding extracellular matrix is unaffected by PDT; thus, tissue healing is excellent and PDT seldom causes scars. This, combined with the ease of light application, has made PDT a popular treatment for cancers and pre-cancerous conditions in human beings. Moreover, because photosensitiser dyes are fluorescent and selectively accumulate in tumour tissues, they can additionally be used to visualise and discriminate tumour from normal tissues, thereby improving the accuracy of tumour surgery. In veterinary practice, PDT has been used successfully for treatment of superficial squamous cell carcinomas of the feline nasal planum; urinary tract, urinary bladder and prostate neoplasia in dogs; and equine sarcoids. The purpose of this article is to provide a comparative review of the current literature on PDT in human and veterinary medicine, and to establish a basis for future development of PDT in veterinary medicine.

Cancer-selective, single agent chemoradiosensitising gold nanoparticles.

Two nanometre gold nanoparticles (AuNPs), bearing sugar moieties and/or thiol-polyethylene glycol-amine (PEG-amine), were synthesised and evaluated for their in vitro toxicity and ability to radiosensitise cells with 220 kV and 6 MV X-rays, using four cell lines representing normal and cancerous skin and breast tissues. Acute 3 h exposure of cells to AuNPs, bearing PEG-amine only or a 50:50 ratio of alpha-galactose derivative and PEG-amine resulted in selective uptake and toxicity towards cancer cells at unprecedentedly low nanomolar concentrations. Chemotoxicity was prevented by co-administration of N-acetyl cysteine antioxidant, or partially prevented by the caspase inhibitor Z-VAD-FMK. In addition to their intrinsic cancer-selective chemotoxicity, these AuNPs acted as radiosensitisers in combination with 220 kV or 6 MV X-rays. The ability of AuNPs bearing simple ligands to act as cancer-selective chemoradiosensitisers at low concentrations is a novel discovery that holds great promise in developing low-cost cancer nanotherapeutics.

Gold nanoparticles for cancer radiotherapy: a review.

Radiotherapy is currently used in around 50% of cancer treatments and relies on the deposition of energy directly into tumour tissue. Although it is generally effective, some of the deposited energy can adversely affect healthy tissue outside the tumour volume, especially in the case of photon radiation (gamma and X-rays). Improved radiotherapy outcomes can be achieved by employing ion beams due to the characteristic energy deposition curve which culminates in a localised, high radiation dose (in form of a Bragg peak). In addition to ion radiotherapy, novel sensitisers, such as nanoparticles, have shown to locally increase the damaging effect of both photon and ion radiation, when both are applied to the tumour area. Amongst the available nanoparticle systems, gold nanoparticles have become particularly popular due to several advantages: biocompatibility, well-established methods for synthesis in a wide range of sizes, and the possibility of coating of their surface with a large number of different molecules to provide partial control of, for example, surface charge or interaction with serum proteins. This gives a full range of options for design parameter combinations, in which the optimal choice is not always clear, partially due to a lack of understanding of many processes that take place upon irradiation of such complicated systems. In this review, we summarise the mechanisms of action of radiation therapy with photons and ions in the presence and absence of nanoparticles, as well as the influence of some of the core and coating design parameters of nanoparticles on their radiosensitisation capabilities.

Engineered neural tissue with aligned, differentiated adipose-derived stem cells promotes peripheral nerve regeneration across a critical sized defect in rat sciatic nerve.

Adipose-derived stem cells were isolated from rats and differentiated to a Schwann cell-like phenotype in vitro. The differentiated cells (dADSCs) underwent self-alignment in a tethered type-1 collagen gel, followed by stabilisation to generate engineered neural tissue (EngNT-dADSC). The pro-regenerative phenotype of dADSCs was enhanced by this process, and the columns of aligned dADSCs in the aligned collagen matrix supported and guided neurite extension in vitro. EngNT-dADSC sheets were rolled to form peripheral nerve repair constructs that were implanted within NeuraWrap conduits to bridge a 15 mm gap in rat sciatic nerve. After 8 weeks regeneration was assessed using immunofluorescence imaging and transmission electron microscopy and compared to empty conduit and nerve graft controls. The proportion of axons detected in the distal stump was 3.5 fold greater in constructs containing EngNT-dADSC than empty tube controls. Our novel combination of technologies that can organise autologous therapeutic cells within an artificial tissue construct provides a promising new cellular biomaterial for peripheral nerve repair.

A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury.

AIM: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success. MATERIALS & METHODS: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control. RESULTS: NCSCs and differentiated Schwann cell-like adipose-derived stem cells did not increase astrocyte reactivity. Highly reactive responses to bone marrow mesenchymal stem cells and Schwann cells were equivalent. CONCLUSION: This approach can screen therapeutic cells prior to in vivo testing, allowing cells likely to trigger a substantial astrocyte response to be identified at an early stage. NCSCs and differentiated Schwann cell-like adipose-derived stem cells may be useful in treating CNS damage without increasing astrogliosis.

Fully protected glycosylated zinc (II) phthalocyanine shows high uptake and photodynamic cytotoxicity in MCF-7 cancer cells.

Phthalocyanine photosensitizers are effective in anticancer photodynamic therapy (PDT) but suffer from limited solubility, limited cellular uptake and limited selectivity for cancer cells. To improve these characteristics, we synthesized isopropylidene-protected and partially deprotected tetra ß-glycosylated zinc (II) phthalocyanines and compared their uptake and accumulation kinetics, subcellular localization, in vitro photocytotoxicity and reactive oxygen species generation with those of disulfonated aluminum phthalocyanine. In MCF-7 cancer cells, one of the compounds, zinc phthalocyanine {4}, demonstrated 10-fold higher uptake, 5-fold greater PDT-induced cellular reactive oxygen species concentration and 2-fold greater phototoxicity than equimolar (9 µm) disulfonated aluminum phthalocyanine. Thus, isopropylidene-protected ß-glycosylation of phthalocyanines provides a simple method of improving the efficacy of PDT.

Engineering an integrated cellular interface in three-dimensional hydrogel cultures permits monitoring of reciprocal astrocyte and neuronal responses.

This study reports a new type of three-dimensional (3D) tissue model for studying interactions between cell types in collagen hydrogels. The aim was to create a 3D cell culture model containing separate cell populations in close proximity without the presence of a mechanical barrier, and demonstrate its relevance to modeling the axon growth-inhibitory cellular interfaces that develop in the central nervous system (CNS) in response to damage. This provides a powerful new tool to determine which aspects of the astroglial scar response and subsequent neuronal regeneration inhibition are determined by the presence of the other cell types. Astrocytes (CNS glia) and dissociated dorsal root ganglia (DRG; containing neurons and peripheral nervous system [PNS] glia) were seeded within collagen solution at 4 °C in adjacent chambers of a stainless steel mould, using cells cultured from wild-type or green fluorescent protein expressing rats, to track specific populations. The divider between the chambers was removed using a protocol that allowed the gels to integrate without mixing of the cell populations. Following setting of the gels, they were maintained in culture for up to 15 days. Reciprocal astrocyte and neuronal responses were monitored using confocal microscopy and 3D image analysis. At DRG:astrocyte interfaces, by 5 days there was an increase in the number of astrocytes at the interface followed by hypertrophy and increased glial fibrillary acidic protein expression at 10 and 15 days, indicative of reactive gliosis. Neurons avoided crossing DRG:astrocyte interfaces, and neuronal growth was restricted to the DRG part of the gel. By contrast, neurons were able to grow freely across DRG:DRG interfaces, demonstrating the absence of a mechanical barrier. These results show that in a precisely controlled 3D environment, an interface between DRG and astrocyte cultures is sufficient to trigger reactive gliosis and inhibition of neuronal regeneration across the interface. Different aspects of the astrocyte response could be independently monitored, providing an insight into the formation of a glial scar. This technology has wide potential for researchers wishing to maintain and monitor interactions between adjacent cell populations in 3D culture.

Regulation of PP2A activity by Mid1 controls cranial neural crest speed and gangliogenesis.

X-linked Opitz syndrome (XLOS), caused by mutation in the MID1 gene, is a midline malformation syndrome with obvious craniofacial abnormalities. Because cranial neural crest cells (CNC) play a pivotal role in cranial morphogenesis, we examined the spatio-temporal expression of cMid1 in chick embryos and investigated if alterations in Mid1 protein function, specifically the ability of Mid1 to negatively regulate levels of protein phosphatase 2A (PP2A), affected CNC survival or migration. During the main phase of CNC migration (stage 9 to 11) cMid1 is strongly expressed within r2 and a subset of CNC in cranial mesenchyme at the level of r1/2 to the isthmus, but is not expressed in more caudal CNC streams. Inhibiting cMid1 function in r2 elevated PP2A levels. Overexpression of PP2A in r2 slowed CNC migration in vitro and in ovo and inhibited trigeminal gangliogenesis. Conversely in r4, forced expression of cMid1, or pharmacological inhibition of PP2A lowered PP2A levels. Inhibition of PP2A in r4 CNC in vitro up-regulated the disintegrin and metalloprotease ADAM10 and selectively increased CNC motility on fibronectin and collagen substrates, but not on laminin. In ovo, inhibiting PP2A activity in r4 increased CNC migration and hastened formation of the geniculate/vestibuloacoustic ganglion, comprising mostly epibranchial placode neuroblasts. Placodal neuroblast migration into the cranial mesenchyme is known to depend on the presence of r4 CNC and we show that inhibition of PP2A in r4 CNC causes premature breakdown of the epibranchial placode basement membrane and early immigration of placodal neuroblasts. In all cases, CNC proliferation and death were unaffected by altered PP2A levels. We propose that factors capable of altering PP2A activity, such as Mid1, affect CNC motility and matrix remodeling, thereby modulating craniofacial development.

Antioxidant inhibitors potentiate the cytotoxicity of photodynamic therapy.

Photodynamic therapy (PDT) is an increasingly popular anticancer treatment that uses photosensitizer, light and tissue oxygen to generate cytotoxic reactive oxygen species (ROS) within illuminated cells. Acting to counteract ROS-mediated damage are various cellular antioxidant pathways. In this study, we combined PDT with specific antioxidant inhibitors to potentiate PDT cytotoxicity in MCF-7 cancer cells. We used disulphonated aluminium phthalocyanine photosensitizer plus various combinations of the antioxidant inhibitors: diethyl-dithiocarbamate (DDC, a Cu/Zn-SOD inhibitor), 2-methoxyestradiol (2-ME, a Mn-SOD inhibitor), l-buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and 3-amino-1,2,4-triazole (3-AT, a catalase inhibitor). BSO, singly or in combination with other antioxidant inhibitors, significantly potentiated PDT cytotoxicity, corresponding with increased ROS levels and apoptosis. The greatest potentiation of cell death over PDT alone was seen when cells were preincubated for 24 h with 300 µM BSO plus 10 mM 3-AT (1.62-fold potentiation) or 300 µM BSO plus 1 µM 2-ME (1.52-fold), or with a combination of all four inhibitors (300 µM BSO, 10 mM 3-AT, 1 µM 2-ME and 10 µM DDC: 1.4-fold). As many of these inhibitors have already been clinically tested, this work facilitates future in vivo studies.

Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit.

After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

Homing of stem cells to sites of inflammatory brain injury after intracerebral and intravenous administration: a longitudinal imaging study.

INTRODUCTION: This study aimed to determine the homing potential and fate of epidermal neural crest stem cells (eNCSCs) derived from hair follicles, and bone marrow-derived stem cells (BMSCs) of mesenchymal origin, in a lipopolysaccharide (LPS)-induced inflammatory lesion model in the rat brain. Both eNCSCs and BMSCs are easily accessible from adult tissues by using minimally invasive procedures and can differentiate into a variety of neuroglial lineages. Thus, these cells have the potential to be used in autologous cell-replacement therapies, minimizing immune rejection, and engineered to secrete a variety of molecules. METHODS: Both eNCSCs and BMSCs were prelabeled with iron-oxide nanoparticles (IO-TAT-FITC) and implanted either onto the corpus callosum in healthy or LPS-lesioned animals or intravenously into lesioned animals. Both cell types were tracked longitudinally in vivo by using magnetic resonance imaging (MRI) for up to 30 days and confirmed by postmortem immunohistochemistry. RESULTS: Transplanted cells in nonlesioned animals remained localized along the corpus callosum. Cells implanted distally from an LPS lesion (either intracerebrally or intravenously) migrated only toward the lesion, as seen by the localized MRI signal void. Fluorescence microscopy of the FITC tag on the nanoparticles confirmed the in vivo MRI data, CONCLUSIONS: This study demonstrated that both cell types can be tracked in vivo by using noninvasive MRI and have pathotropic properties toward an inflammatory lesion in the brain. As these cells differentiate into the glial phenotype and are derived from adult tissues, they offer a viable alternative autologous stem cell source and gene-targeting potential for neurodegenerative and demyelinating pathologies.

Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence.

To be effective for tissue repair, satellite cells (the stem cells of adult muscle) must survive the initial activation from quiescence. Using an in vitro model of satellite cell activation, we show that erbB1, erbB2 and erbB3, members of the EGF receptor tyrosine kinase family, appear on satellite cells within 6 h of activation. We show that signalling via erbB2 provides an anti-apoptotic survival mechanism for satellite cells during the first 24 h, as they progress to a proliferative state. Inhibition of erbB2 signalling with AG825 reduced satellite cell numbers, concomitant with elevated caspase-8 activation and TUNEL labelling of apoptotic satellite cells. In serum-free conditions, satellite cell apoptosis could be largely prevented by a mixture of erbB1, erbB3 and erbB4 ligand growth factors, but not by neuregulin alone (erbB3/erbB4 ligand). Furthermore, using inhibitors specific to discrete intracellular signalling pathways, we identify MEK as a pro-apoptotic mediator, and the erbB-regulated factor STAT3 as an anti-apoptotic mediator during satellite cell activation. These results implicate erbB2 signalling in the preservation of a full compliment of satellite cells as they activate in the context of a damaged muscle.

Heparin-binding EGF-like growth factor shows transient left-right asymmetrical expression in mouse myotome pairs.

Heparin-binding EGF-like growth factor (HB-EGF) is a potent mitogen and chemoattractant for diverse cell types including, keratinocytes, fibroblasts and vascular smooth muscle cells. In adult mice, skeletal muscle and endothelial cells prominently express HB-EGF, although analysis of embryonic expression has been limited to studies of heart and kidney development. Here we survey HB-EGF mRNA expression in E7.5-E15 mouse embryos and show that HB-EGF is expressed in branchial arches, limb buds and, transiently, in mature somites between E9.25 and E11. This somitic expression is restricted to the myotomal compartment. Intriguingly, within myotome pairs, the expression of HB-EGF is stronger on the left side of the body, whilst cognate receptors, ErbB1 and ErbB4, are symmetrically expressed in left and right somite pairs. In iv/iv mutant embryos, with inverted left-right body axis, the expression of HB-EGF was also inverted, now being stronger in myotomes on the right side of the body. Thus, the expression of HB-EGF in myotome pairs is regulated by global cues that define the left-right body axis.

Mouse myotomes pairs exhibit left-right asymmetric expression of MLC3F and alpha-skeletal actin.

Most muscle originates from the myotomal compartment of the somites, paired structures flanking the neural tube. Whereas vertebrate embryos show molecular and morphological asymmetry about the left-right body axis, somitic myogenesis is thought to occur symmetrically. Here, we provide the first evidence that myotome pairs are transiently left-right asymmetric, with higher expression of alpha-skeletal actin and myosin light chain 3F (MLC3F) on the left side between embryonic day 9.5-10.25. In iv mutants with situs inversus, the asymmetric expression of alpha-skeletal actin and MLC3F was inverted, showing that this process is regulated by global left-right axis cues, initiated before gastrulation. However, although left-sided identity is later maintained by Pitx2 genes, we found that Pitx2c null embryos have normal left-biased expression of alpha-skeletal actin and MLC3F. Myotome asymmetry, therefore, is downstream of the iv mutation but upstream of, or unrelated to, the Pitx2c pathway.

Myf5 expression in satellite cells and spindles in adult muscle is controlled by separate genetic elements.

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5(nlacZ/+) mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.

Muscle satellite cells adopt divergent fates: a mechanism for self-renewal?

Growth, repair, and regeneration of adult skeletal muscle depends on the persistence of satellite cells: muscle stem cells resident beneath the basal lamina that surrounds each myofiber. However, how the satellite cell compartment is maintained is unclear. Here, we use cultured myofibers to model muscle regeneration and show that satellite cells adopt divergent fates. Quiescent satellite cells are synchronously activated to coexpress the transcription factors Pax7 and MyoD. Most then proliferate, down-regulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from immediate differentiation. These cells are typically located in clusters, together with Pax7-ve progeny destined for differentiation. Some of the Pax7+ve/MyoD-ve cells then leave the cell cycle, thus regaining the quiescent satellite cell phenotype. Significantly, noncycling cells contained within a cluster can be stimulated to proliferate again. These observations suggest that satellite cells either differentiate or switch from terminal myogenesis to maintain the satellite cell pool.

Roles of erbB4, rhombomere-specific, and rhombomere-independent cues in maintaining neural crest-free zones in the embryonic head.

Within the developing vertebrate head, the migration of neural tube-derived neural crest cells (NCCs) through the cranial mesenchyme is patterned into three streams, with mesenchyme adjacent to rhombomeres (r)3 and r5 maintained NCC-free. The receptor tyrosine kinase erbB4 is expressed within r3 and r5 and is required to maintain the r3-adjacent NCC-free zone in mouse embryos. In this study, we demonstrate that the extent of r3 involvement in patterning mouse NCC migration is restricted to the same dorsolateral region regulated by erbB4. In chick embryos, we show that erbB4 signaling similarly maintains the r3-adjacent NCC-free zone. However, although r5 expresses erbB4, this is insufficient to maintain the r3-adjacent NCC-free zone in grafting experiments where r5 replaced r3, indicating that erbB4 requires additional factors at the A-P level of r3 to pattern NCC migration. Furthermore, we show that the r5-adjacent NCC-free zone is maintained independently of r5, but requires surface ectoderm. Finally, we demonstrate that avian cranial surface ectoderm is patterned molecularly, with dorsolateral surface ectoderm at the levels of r2/3 and r7 expressing the sulfatase QSulf1 in quail, or the orthologue CSulf1 in chick. Aberrant NCC migration into r3-adjacent mesenchyme correlated with more focused QSulf1 expression in r2/3 surface ectoderm.