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Fibroblast growth factors (FGFs) are diffusible polypeptides released by a variety of cell types. FGF8 subfamily members regulate embryonic development processes through controlling progenitor cell growth and differentiation, and are also functional in adults in tissue repair to maintain tissue homeostasis. FGF8 family members exhibit unique binding affinities with FGF receptors and tissue distribution patterns. Increasing evidence suggests that, by regulating multiple cellular signaling pathways, alterations in the FGF8 subfamily are involved in craniofacial development, odontogenesis, tongue development and salivary gland branching morphogenesis. Aberrant FGF signaling transduction, caused by mutations as well as abnormal expression or isoform splicing, plays an important role in the development of oral diseases. Targeting FGF8 subfamily members provides a new promising strategy for the treatment of oral diseases. The aim of this review was to summarize the aberrant regulations of FGF8 subfamily members and their potential implications in oral-maxillofacial diseases.

Organoids generating major cortical cell types in distinct compartments are used to study cortical development, evolution and disorders. However, the lack of morphogen gradients imparting cortical positional information and topography in current systems hinders the investigation of complex phenotypes. Here, we engineer human cortical assembloids by fusing an organizer-like structure expressing fibroblast growth factor 8 (FGF8) with an elongated organoid to enable the controlled modulation of FGF8 signaling along the longitudinal organoid axis. These polarized cortical assembloids mount a position-dependent transcriptional program that in part matches the in vivo rostrocaudal gene expression patterns and that is lost upon mutation in the FGFR3 gene associated with temporal lobe malformations and intellectual disability. By producing spatially oriented cell populations with signatures related to frontal and temporal area identity within individual assembloids, this model recapitulates in part the early transcriptional divergence embedded in the protomap and enables the study of cortical area-relevant alterations underlying human disorders. Cortical development is influenced by morphogen gradients. To mimic patterning events during brain development, polarized cortical assembloids are generated with the help of a localized FGF8 source.

Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant. How this temperature independence is achieved is a mystery that we address in this study. Using RT-qPCR we show that the endogenous fgf8 mRNA concentration decreases during somitogenesis and correlates with the exponent of the shrinking pre-somitic mesoderm (PSM) size. As the temperature decreases, the dynamics of fgf8 and many other gene transcripts, as well as the segmentation frequency and the PSM shortening and tail growth rates slows down as T–T c (with T c  = 14.4 °C). This behavior characteristic of a system near a critical point may account for the temperature independence of somitogenesis in zebrafish. In Zebrafish, the dynamics of fgf8 and other gene transcripts as well as segmentation frequency, shortening of pre-somitic mesoderm and tail growth rate slows down with lower temperature. This may explain the temperature independence of somitogenesis.

Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome. CHARGE syndrome is a rare genetic condition that causes various developmental abnormalities, including heart defects, deafness and neurological defects. In most cases, it is caused by mutations in a human gene called CHD7. CHD7 is known to control the expression of other genes during embryonic development, but the molecular mechanisms by which mutations in CHD7 lead to the neural defects found in CHARGE syndrome are unclear. During embryonic development, the neural tube—the precursor to the nervous system—is divided into segments, which give rise to different neural structures. The r1 segment, for example, forms the cerebellum, and the secretion of a protein called FGF8 (short for fibroblast growth factor 8) by a nearby structure called the isthmus organiser has an important role in this process. Since a reduction in FGF8 causes defects similar to those found in CHARGE syndrome, Yu et al. decided to investigate if the FGF signalling pathway was involved in this syndrome. Mice should have two working copies of the Chd7 gene, and mice that lack one of these suffer from symptoms similar to those of humans with CHARGE syndrome. Yu et al. examined the embryos of these mice and found that the isthmus organiser produced less FGF8. Embryos with no working copies of the gene completely lost the r1 segment. The loss of this segment appeared to be caused by changes in the expression of homeobox genes (the genes that determine the identity of brain segments). Embryos that did not have any working copies of the Chd7 gene died early in development, which made further studies impossible. However, embryos that had one working copy of the Chd7 gene survived, and Yu et al. took advantage of this to study the effects of reduced FGF8 expression on these mice. These experiments showed that mice with just one working copy of the Fgf8 gene and one working copy of the Chd7 gene had a small cerebellar vermis. This part of the cerebellum is known to be very sensitive to changes in FGF8 signalling. Yu et al. then used an MRI scanner to look at the cerebellar vermis in patients with CHARGE syndrome, and found that more than half of the patients had abnormal cerebella. In addition to confirming that studies on mouse embryos can provide insights into human disease, the work of Yu et al. add defects in the cerebellar vermis to the list of developmental abnormalities associated with CHARGE syndrome. The next step will be to test if any mutations in the human FGF8 gene can contribute to cerebellar defects in CHARGE syndrome, and to investigate if any other developmental defects in CHARGE syndrome are associated with abnormal FGF8 levels.

The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4 , 8 , 9 , and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex. Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex. Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex. Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex. Fgf8 expression. We found that Amex. Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex. Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex. Fgf8 expression seen in the axolotl.

Fibroblast growth factors in development The developing limb bud possesses a small ridge, the apical ectodermal ridge (AER), that produces signals controlling development of the limb along the proximal–distal axis (from the upper arms to the finger tips). Fibroblast growth factors (FGFs) are known to be key AER signals, but as four FGFs are expressed in the mouse AER, it has been difficult to understand their roles. Mariani et al . used genetic techniques to delete different combinations of FGFs from the mouse limb, thereby revealing the contribution made by each FGF to the total AER-FGF signal. Only one of the four AER-FGFs, Fgf8 , was found to be essential for normal limb development. This dispels a longstanding notion that there is a positive feedback loop between the three other FGF genes expressed in the posterior AER and the sonic hedgehog gene. They also provide the first genetic evidence that the AER-FGFs serve as distalizing factors for establishing limb patterning, suggesting a role of FGFs as patterning molecules. They present a model that synthesizes the new findings with several other controversial papers published in recent years on the validity of the 'progress zone' versus the 'early specification' model of limb development. Genetic techniques have been used to delete different combinations of fibroblast growth factors (FGFs) from the mouse limb, to study the contribution that each FGF makes to the total apical ectodermal ridge (AER)–FGF signal. Out of the four AER–FGFs, it is shown that only one of them, Fgf8 is sufficient for normal limb development. This dispels a longstanding notion that there is a positive feedback loop between the three other FGF genes expressed in the posterior AER, and the sonic hedgehog gene. Half a century ago, the apical ectodermal ridge (AER) at the distal tip of the tetrapod limb bud was shown to produce signals necessary for development along the proximal–distal (P–D) axis, but how these signals influence limb patterning is still much debated 1 , 2 . Fibroblast growth factor (FGF) gene family members are key AER-derived signals 3 , 4 , with Fgf4 , Fgf8 , Fgf9 and Fgf17 expressed specifically in the mouse AER 5 . Here we demonstrate that mouse limbs lacking Fgf4, Fgf9 and Fgf17 have normal skeletal pattern, indicating that Fgf8 is sufficient among AER-FGFs to sustain normal limb formation. Inactivation of Fgf8 alone causes a mild skeletal phenotype 6 , 7 ; however, when we also removed different combinations of the other AER-FGF genes, we obtained unexpected skeletal phenotypes of increasing severity, reflecting the contribution that each FGF can make to the total AER-FGF signal. Analysis of the compound mutant limb buds revealed that, in addition to sustaining cell survival, AER-FGFs regulate P–D-patterning gene expression during early limb bud development, providing genetic evidence that AER-FGFs function to specify a distal domain and challenging the long-standing hypothesis that AER-FGF signalling is permissive rather than instructive for limb patterning. We discuss how a two-signal model for P–D patterning can be integrated with the concept of early specification to explain the genetic data presented here.

The morphogen FGF8 establishes graded positional cues imparting regional cellular responses via modulation of early target genes. The roles of FGF signaling and its effector genes remain poorly characterized in human experimental models mimicking early fetal telencephalic development. We used hiPSC-derived cerebral organoids as an in vitro platform to investigate the effect of FGF8 signaling on neural identity and differentiation. We found that FGF8 treatment increases cellular heterogeneity, leading to distinct telencephalic and mesencephalic-like domains that co-develop in multi-regional organoids. Within telencephalic regions, FGF8 affects the anteroposterior and dorsoventral identity of neural progenitors and the balance between GABAergic and glutamatergic neurons, thus impacting spontaneous neuronal network activity. Moreover, FGF8 efficiently modulates key regulators responsible for several human neurodevelopmental disorders. Overall, our results show that FGF8 signaling is directly involved in both regional patterning and cellular diversity in human cerebral organoids and in modulating genes associated with normal and pathological neural development. Healthy brain development in the human embryo relies on the precise coordination of numerous molecular signals that guide the formation of distinct brain regions in their correct locations. Molecules that diffuse through embryonic tissues, known as morphogens, serve as spatial and temporal cues that help cells determine their position within the developing brain. These positional signals are crucial for the proper formation of specific brain regions along the embryo’s principal axes. FGF8 is a well-characterized morphogen that influences the anterior-to-posterior regional identity of brain cells in model organisms such as mice. However, studying this process in human embryos poses both technical and ethical challenges, meaning that little is known about the molecular bases of how developing brain cells determine their position along different axes. Understanding these molecular mechanisms is essential for gaining insights into human brain function and the origins of neurodevelopmental disorders. Bertacchi et al. developed a new 'organ-in-a-dish' system – also known as an organoid – using human induced pluripotent stem cells. The research team combined 2D cell cultures on flat surfaces with 3D tissue culture techniques to create a more reproducible cerebral organoid protocol. This approach enabled the investigation of the role of FGF8 in human brain development within a controlled laboratory setting. Treatment with FGF8 enhanced brain cell diversity, as measured by gene expression analysis through single-cell RNA sequencing. Notably, distinct regions resembling the forebrain (telencephalon) and the midbrain (mesencephalon) emerged in FGF8-treated organoids. Within the telencephalic region, the cell type composition shifted, favoring neurons typically found in the ventral (lower) parts of the human brain. This altered the activity of the neural network, as evidenced by direct electrical signal measurements. Overall, Bertacchi et al. demonstrated that a single molecular signal, FGF8, can drive the formation of distinct brain regions along multiple axes in human brain organoids. They also identified genes regulated by FGF8 that are associated with neurodevelopmental disorders. One such gene, NR2F1, is well-studied for its involvement in conditions such as intellectual disability, autism and epilepsy. This work provides a biologically accurate cell culture model, offering a valuable tool for advancing research into human brain development and associated neurological diseases.

Key Points Fibroblast growth factor (FGF) signalling controls a myriad of processes in embryonic development and in tissue homeostasis and metabolism in the adult. Recent structural studies have provided a glimpse of the complexity of molecular control that is in place to fine-tune this signalling system to enable it to produce specific signalling outputs in diverse biological contexts. The interaction of FGFs with heparan sulphate glycosaminoglycan chains of heparan sulphate proteoglycans in the pericellular and extracellular matrix defines their mode of action, that is, whether an FGF acts in a paracrine or endocrine fashion. It also determines the shape of gradient formed by a paracrine FGF ligand in the extracellular matrix, which in turn is a determinant of the biological response to that ligand. In addition to mechanisms common to all FGFs, such as the interaction with heparan sulphate, the biological activity of individual ligands or ligand subfamilies is regulated by mechanisms unique to these ligands: amino-terminal alternative splicing controls the activity of FGF8 subfamily ligands; homodimerization autoinhibits the activity of FGF9 subfamily ligands; and site-specific proteolytic cleavage inactivates the phosphaturic hormone FGF23. Alternative splicing in the extracellular immunoglobulin-like domain 3 (D3) of FGF receptor 1 (FGFR1), FGFR2 and FGFR3 primarily determines the ligand-binding specificity of these receptors. This splicing event is fundamental to the establishment of directional paracrine FGF signalling between the epithelium and the mesenchyme, which underlies the coordinated cellular processes that govern organ development. Klotho co-receptors convert FGFRs into specific receptors for endocrine FGFs by a dual mechanism; these co-receptors not only enhance the binding affinity of FGFRs for endocrine FGFs but concomitantly suppress the binding of paracrine FGFs to FGFRs. The finding that heparan sulphate is dispensable for signalling by endocrine FGFs implies that Klotho co-receptors also promote FGFR dimerization upon endocrine FGF binding, which is required for FGFR activation. The structural findings suggest that there may be no functional redundancy among FGF ligands, and genetic data support this conclusion. Hence, future studies should concentrate on identifying novel ligand-specific functions of FGF signalling. Structural data has provided insight into the molecular mechanisms that modulate fibroblast growth factor (FGF) signalling to generate distinct biological outputs in development, tissue homeostasis and metabolism. Mechanisms include alternative splicing of ligand and receptor, homodimerization and site-specific proteolytic cleavage of ligand, and interaction of ligand and receptor with heparan sulphate and Klotho co-receptors. Fibroblast growth factors (FGFs) mediate a broad range of functions in both the developing and adult organism. The accumulated wealth of structural information on the FGF signalling pathway has begun to unveil the underlying molecular mechanisms that modulate this system to generate a myriad of distinct biological outputs in development, tissue homeostasis and metabolism. At the ligand and receptor level, these mechanisms include alternative splicing of the ligand (FGF8 subfamily) and the receptor (FGFR1–FGFR3), ligand homodimerization (FGF9 subfamily), site-specific proteolytic cleavage of the ligand (FGF23), and interaction of the ligand and the receptor with heparan sulphate cofactor and Klotho co-receptor.

Human fibroblast growth factor 8b (FGF8b) was expressed based on a baculovirus expression vector system (BEVS) and identified as having a protective effect on Parkinson’s disease. Immunoblotting demonstrated that rhFGF8b proteins were recognized by a human anti-FGF8b antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield and protein quality. Our results indicated that the rhFGF8b was first detectable at 36 h postinfection and reached a maximum at 60 h. A multiplicity of infection (MOI) of 8 pfu/mL was suitable for harvest. The target protein was purified by heparin-affinity chromatography. In vitro methylthiazol tetrazolium (MTT) assays demonstrated that the purified rhFGF8b could significantly stimulate proliferation of NIH3T3 cells. Furthermore, to elucidate the effect of rhFGF8b on Parkinson’s disease, we used FGF8b pretreatment on a cell model of Parkinson’s disease. The results indicated that rhFGF8b prevented necrosis and apoptosis of 1-METHYL-4-phenyl pyridine (MPP + ) treated PC12 cells. Moreover, the effect of FGF8b on messenger RNA (mRNA) levels of apoptosis and ERS genes was investigated to clarify the molecular mechanisms of FGF8b. The results suggest that FGF8b exerts neuroprotective effects by alleviating endoplasmic reticulum (ER) stress during PD. These results suggest that FGF8b may be a promising candidate therapeutic drug for neurodegenerative diseases related to ER stress.

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage. Developmental genes are often regulated by multiple elements, yet their relative contribution to gene expression remains poorly understood. Here the authors apply in vivo CRISPR/Cas9 genome engineering to find two distinct regulatory logics directing Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary.