The role of Fgf8 in telencephalic and diencephalic patterning

Correct patterning of the developing brain is crucial importance for accurate wiring and function. Although the adult brain contains many complex structures, it begins with a simple structure—the neural tube. As it develops, the neural tube is divided into several regions, including the telencephalon, diencephalon, midbrain, and hindbrain. In each of these regions, signaling molecules are secreted from discrete zones, which establish positional information and regulate regional growth. There are many mechanistic questions that remain to be resolved about the action of these growth and differentiation factors. The cellular factors mediating patterning in response to these factors are largely unknown. Furthermore, identical differentiation factors are expressed in different regions of the brain and yet control significantly different patterning mechanisms, and the factors that control region-specific responses to these factors are mostly obscure. Furthermore, differentiation factors also show dramatically different expression patterns in different vertebrate species that may underlie changes in brain structure, but the mechanisms by which these changes in gene expression occur poorly understood. To address these issues, we discuss the role of Fgf8, which controls anterior/posterior patterning in different regions of the developing brain. We also discuss how modifications of Fgf8 expression in the diencephalon controlled by retrotransposons can change the shape and function of the brain in various species.     

Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development

Complex spatiotemporal expression patterns of fgf3 and fgf8 within the developing zebrafish forebrain suggest their involvement in its regionalisation and early development. These factors have unique and combinatorial roles during development of more posterior brain regions, and here we report similar findings for the developing forebrain. We show that Fgf8 and Fgf3 regulate different aspects of telencephalic development, and that Fgf3 alone is required for the expression of several telencephalic markers. Within the diencephalon, Fgf3 and Fgf8 act synergistically to pattern the ventral thalamus, and are implicated in the regulation of optic stalk formation, whereas loss of Fgf3 alone results in defects in ZLI development. Forebrain commissure formation was abnormal in the absence of either Fgf3 or Fgf8; however, most severe defects were observed in the absence of both. Defects were observed in patterning of both the midline territory, within which the commissures normally form, and neuronal populations, whose axons comprise the commissures. Analysis of embryos treated with an FGFR inhibitor suggests that continuous FGF signalling is required from gastrulation stages for normal forebrain patterning, and identifies additional requirements for FGFR activity.     

Distinct functions of the major Fgf8 spliceform, Fgf8b, before and during mouse gastrulation

The vertebrate Fgf8 gene produces multiple protein isoforms by alternative splicing. Two evolutionarily conserved spliceforms, Fgf8a and Fgf8b, exhibit distinct bioactivities, with Fgf8b having a more potent inductive activity due to higher affinity for Fgf receptors. To investigate the in vivo requirement for Fgf8b, we created a splice-site mutation abolishing Fgf8b expression in mice. Analysis of this mutant has uncovered a novel function of Fgf8 signaling before the onset of gastrulation. We show that the loss of Fgf8b disrupts the induction of the brachyury gene in the pregastrular embryo and, in addition, disrupts the proper alignment of the anteroposterior axis with the shape of the embryo and the uterine axes at embryonic day (E) 6.5. Importantly, Fgf8-null embryos display the same phenotype as Fgf8b-deficient embryos at E6.5, demonstrating that signaling by Fgf8b is specifically required for development of the pregastrular embryo. By contrast, during gastrulation, Fgf8a can partially compensate for the loss of Fgf8b in mesoderm specification. We show that an increased level of Fgf8a expression, which leads to Fgf4 expression in the primitive streak, can also promote mesoderm migration in the absence of Fgf8b. Therefore, different Fgf signals may have distinct requirements for the morphogenesis and gene regulation before and during gastrulation. Importantly, our findings implicate Fgf8 in the morphogenetic process that establishes the defined relationship between the axes of the embryo and the uterus at the beginning of gastrulation, a perplexing phenomenon discovered two decades ago.     

Fgf8 and Fgf3 are required for zebrafish ear placode induction,maintenance and inner ear patterning

The vertebrate inner ear develops from initially 'simple' ectodermal placode and vesicle stages into the complex three-dimensional structure which is necessary for the senses of hearing and equilibrium. Although the main morphological events in vertebrate inner ear development are known, the genetic mechanisms controlling them are scarcely understood. Previous studies have suggested that the otic placode is induced by signals from the chordamesoderm and the hindbrain, notably by fibroblast growth factors (Fgfs) and Wnt proteins. Here we study the role of Fgf8 as a bona-fide hindbrain-derived signal that acts in conjunction with Fgf3 during placode induction, maintenance and otic vesicle patterning. Acerebellar (ace) is a mutant in the fgf8 gene that results in a non-functional Fgf8 product. Homozygous mutants for acerebellar (ace) have smaller ears that typically have only one otolith, abnormal semi-circular canals, and behavioral defects. Using gene expression markers for the otic placode, we find that ace/fgf8 and Fgf-signaling are required for normal otic placode formation and maintenance. Conversely, misexpression of fgf8 or Fgf8-coated beads implanted into the vicinity of the otic placode can increase ear size and marker gene expression, although competence to respond to the induction appears restricted. Cell transplantation experiments and expression analysis suggest that Fgf8 is required in the hindbrain in the rhombomere 4-6 area to restore normal placode development in ace mutants, in close neighbourhood to the forming placode, but not in mesodermal tissues. Fgf3 and Fgf8 are expressed in hindbrain rhombomere 4 during the stages that are critical for placode induction. Joint inactivation of Fgf3 and Fgf8 by mutation or antisense-morpholino injection causes failure of placode formation and results in ear-less embryos, mimicking the phenotype we observe after pharmacological inhibition of Fgf-signaling. Fgf8 and Fgf3 together therefore act during induction and differentiation of the ear placode. In addition to the early requirement for Fgf signaling, the abnormal differentiation of inner ear structures and mechanosensory hair cells in ace mutants, pharmacological inhibition of Fgf signaling, and the expression of fgf8 and fgf3 in the otic vesicle demonstrate independent Fgf function(s) during later development of the otic vesicle and lateral line organ. We furthermore addressed a potential role of endomesomerm by studying mzoep mutant embryos that are depleted of head endomesodermal tissue, including chordamesoderm, due to a lack of Nodal-pathway signaling. In these embryos, early placode induction proceeds largely normally, but the ear placode extends abnormally to midline levels at later stages, suggesting a role for the midline in restricting placode development to dorsolateral levels. We suggest a model of zebrafish inner ear development with several discrete steps that utilize sequential Fgf signals during otic placode induction and vesicle patterning.     

Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea

The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate (PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them potential targets for regenerative therapies.     

Retinoic acid affects craniofacial patterning by changing Fgf8 expression in the pharyngeal ectoderm

Retinoic acid signaling plays important roles in establishing normal patterning and cellular differentiation during embryonic development. In this study, we show that single administration of retinoic acid at embryonic day 8.5 causes homeotic transformation of the lower jaw into upper jaw-like structures. This homeosis was preceded by downregulation of Fgf8 and Sprouty expression in the proximal domain of the first pharyngeal arch. Downregulation of mesenchymal genes such as Dlx5, Hand2, Tbx1 and Pitx2 was also observed. The oropharynx in retinoic acid-treated embryos was severely constricted. Consistent with this observation, Patched expression in the arch endoderm and mesenchyme was downregulated. Thus, retinoic acid affects the expression of subsets of epithelial and mesenchymal genes, possibly disrupting the regional identity of the pharyngeal arch.     

Temporal requirement for hedgehog signaling in ventral telencephalic patterning

Hedgehog signaling is required for multiple aspects of brain development, including growth, the establishment of both dorsal and ventral midline patterning and the generation of specific cell types such as oligodendrocytes and interneurons. To identify more precisely when during development hedgehog signaling mediates these events, we directed the removal of hedgehog signaling within the brain by embryonic day 9 of development, using a FoxG1(Cre) driver line to mediate the removal of a conditional smoothened null allele. We observed a loss of ventral telencephalic patterning that appears to result from an initial lack of specification of these structures rather than by changes in proliferation or cell death. A further consequence of the removal of smoothened in these mice is the near absence of both oligodendrocytes and interneurons. Surprisingly, the dorsal midline appears to be patterned normally in these mutants. Together with previous analyses, the present results demonstrate that hedgehog signaling in the period between E9.0 and E12 is essential for the patterning of ventral regions and the generation of cell types that are thought to largely arise from them.     

Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon

Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.     

Metabolic activity of pigeon thalamic and telencephalic auditory centers

Distribution of activity of mitochondrial oxidative enzyme cytochrome oxidase (CO) was studied in the thalamic (Ov) and telencephalic (field L) auditory centers of the pigeon Columbia livia. Different levels of CO activity are found in the core and belt of the centers: the high CO activity in the core of Ov (nCe) and telencephalic field L2 and the much lower or absent in the peripheral regions (Ovl, Ovm, SPO and L1 and L3). Comparison of our data with those of various avian and reptile species confirms the concept of the common plan of rostral auditory centers in sauropsid amniotes by the principle of the center-periphery (core-belt), which is characteristic of the corresponding mammalian centers. The separation of the central and peripheral parts of these centers is better pronounced in birds than in reptiles.     

Regulation of avian cardiogenesis by Fgf8 signaling

The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis.     

Dose-dependent effects of endotoxin on neurobehavioral functions in humans

Clinical and experimental evidence document that inflammation and increased peripheral cytokine levels are associated with depression-like symptoms and neuropsychological disturbances in humans. However, it remains unclear whether and to what extent cognitive functions like memory and attention are affected by and related to the dose of the inflammatory stimulus. Thus, in a cross-over, double-blind, experimental approach, healthy male volunteers were administered with either placebo or bacterial lipopolysaccharide (LPS) at doses of 0.4 (n = 18) or 0.8 ng/kg of body weight (n = 16). Pro- and anti-inflammatory cytokines, norephinephrine and cortisol concentrations were analyzed before and 1, 1.75, 3, 4, 6, and 24 h after injection. In addition, changes in mood and anxiety levels were determined together with working memory (n-back task) and long term memory performance (recall of emotional and neutral pictures of the International Affective Picture System). Endotoxin administration caused a profound transient physiological response with dose-related elevations in body temperature and heart rate, increases in plasma interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α and IL-1 receptor antagonist (IL-1ra), salivary and plasma cortisol, and plasma norepinephrine. These changes were accompanied by dose-related decreased mood and increased anxiety levels. LPS administration did not affect accuracy in working memory performance but improved reaction time in the high-dose LPS condition compared to the control conditon. In contrast, long-term memory performance was impaired selectively for emotional stimuli after administration of the lower but not of the higher dose of LPS. These data suggest the existence of at least two counter-acting mechanisms, one promoting and one inhibiting cognitive performance during acute systemic inflammation.     

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.     

Fgf8 controls regional identity in the developing thalamus

The vertebrate thalamus contains multiple sensory nuclei and serves as a relay station to receive sensory information and project to corresponding cortical areas. During development, the progenitor region of the diencephalon is divided into three parts, p1, p2 (presumptive thalamus) and p3, along its longitudinal axis. Besides the local expression of signaling molecules such as sonic hedgehog (Shh), Wnt proteins and Fgf8, the patterning mechanisms of the thalamic nuclei are largely unknown. Using mouse in utero electroporation to overexpress or inhibit endogenous Fgf8 at the diencephalic p2/p3 border, we revealed that it affected gene expression only in the p2 region without altering overall diencephalic size or the expression of other signaling molecules. We demonstrated that two distinctive populations in p2, which can be distinguished by Ngn2 and Mash1 in early embryonic diencephalon, are controlled by Fgf8 activity in complementary manner. Furthermore, we found that FGF activity shifts thalamic sensory nuclei on the A/P axis in postnatal brain. Moreover, gene expression analysis demonstrated that FGF signaling shifts prethalamic nuclei in complementary manner to the thalamic shift. These findings suggest conserved roles of FGF signaling in patterning along the A/P axis in CNS, and reveal mechanisms of nucleogenesis in the developing thalamus.     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.