Lens and retina formation require expression of Pitx3 in Xenopus pre-lens ectoderm.

Pitx3 is expressed in tissues fated to contribute to eye development, namely, neurula stage ectoderm and pre-chordal mesoderm, then presumptive lens ectoderm, placode, and finally lens. Pitx3 overexpression alters lens, optic cup, optic nerve, and diencephalon development. Many of the induced anomalies are attributable to midline deficits; however, as assessed by molecular markers, ectopic Pitx3 appears to temporarily enlarge the lens field. These changes are usually insufficient to generate either ectopic lenses to enlarge the eye that eventually differentiates. Conversely, use of a repressor chimera or of antisense morpholinos alters early expression of marker genes, and later inhibits lens development, thereby abrogating retinal induction. Reciprocal grafting experiments using wild-type and morpholino-treated tissues demonstrate that Pitx3 expression in the presumptive lens ectoderm is required for lens formation. Contradictory to recent assertions that retina can form in the absence of a lens, the expression of Pitx3 in the presumptive lens ectoderm is critical for retina development.     

FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation

The ophthalmic trigeminal (opV) placode gives rise exclusively to sensory neurons of the peripheral nervous system, providing an advantageous model for understanding neurogenesis. The signaling pathways governing opV placode development have only recently begun to be elucidated. Here, we investigate the fibroblast growth factor receptor‐4 (FGFR4), an opV expressed gene, to examine if and how FGF signaling regulates opV placode development. After inhibiting FGFR4, Pax3+ opV placode cells failed to delaminate from the ectoderm and did not contribute to the opV ganglion. Blocking FGF signaling also led to a loss of the early and late neuronal differentiation markers Ngn2, Islet‐1, NeuN, and Neurofilament. In addition, without FGF signaling, cells that stalled in the ectoderm lost their opV placode‐specific identity by down‐regulating Pax3. We conclude that FGF signaling, through FGFR4, is necessary for delamination and differentiation of opV placode cells. Developmental Dynamics 238:1073–1082, 2009. © 2009 Wiley‐Liss, Inc.     

FGF8 initiates inner ear induction in chick and mouse

In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermal Fgf10 acting redundantly with neural Fgf3 is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, we took advantage of the unique strengths of the two model organisms. We show that endoderm is necessary for otic induction in the chick and that Fgf8, expressed in the chick endoderm subjacent to Fgf19, is both sufficient and necessary for the expression of Fgf19 in the mesoderm. In the mouse, Fgf8 is also expressed in endoderm as well as in other germ layers in the periotic placode region. We show that otic induction fails in embryos null for Fgf3 and hypomorphic for Fgf8 and expression of mesodermal Fgf10 is reduced. Thus, Fgf8 plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse.     

Wnt10a is involved in AER formation during chick limb development.

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/beta-catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of beta-catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/beta-catenin signaling pathway.     

Expression of mouse Foxi class genes in early craniofacial development.

Recent models of craniofacial development suggest the existence of a common pan-placodal domain lying next to the neural plate, from which all sensory placodes will arise. In support of this idea, several genes are expressed in the surface ectoderm of the head adjacent to the neural plate, before the appearance of genes in specific cranial placodes. In this study, we examine the expression patterns of the mouse Foxi class genes from embryonic day 6.5 to 10.5. Foxi2 is expressed throughout the cranial ectoderm adjacent to the neural plate from the 4-somite stage, later becoming excluded from the otic placode. Foxi3 is expressed in a broad region of the pan-placodal ectoderm adjacent to the neural plate from embryonic day (E) 6.75 to the first somite stage. Its expression becomes restricted to the ectoderm and the endoderm of the branchial pouches at E10.5. Foxi1 expression is first detected in the endolymphatic duct in the otic vesicle at E10.5. These results suggest that the mouse Foxi class genes may play important roles, both during cranial placode specification and in later development of individual cranial sensory structures and other organs derived from the cranial ectoderm.     

Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis

Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.     

Development of the embryonic chick otic placode. I. Light microscopic analysis

At the 7–8 somite stage of embryonic chick development (29-31 hours of incubation), a slightly elliptical island of thickened ectoderm appears laterally on either side of the most distal constricture of the rhombencephalon at the level of the anterior intestinal portal. The appearance and extent of this auditory placode is precisely correlated with the subjacent accumulation of neural crest cells. By 33 hours of incubation, there is a distinct depression in the developing otic placode, and by 40 to 45 hours, the placode is visibly invaginated, forming an epithelial vesicle or otocyst. Carefully staged embryos were serially sectioned, and the area underlying the developing otic placode was traced with a planimeter. It was found that placode size (area 60,000 μm²) is nearly unchanged from 30 to 42 hours of development. During this time interval, the placode cells first become columnar, show nuclear orientation, and then pseudostratify. The increase in placode cell number during this time interval is not likely to be the result of localized, accelerated cell division: the population doubling time of placode cells is eight and one-half hours and the mitotic index of 2.5% is similar to that of cells in an equivalent area of adjacent, non-placode forming head ectoderm. A model of otic placode formation is proposed which suggests that by 30 hours of development, a discrete population of placode forming cells is segregated from head ectoderm. Subsequent epithelial pseudostratification results from accumulation of this dividing population within the limits of the placode.     

Hereditary hair loss and the ancient signaling pathways that regulate ectodermal appendage formation

All epidermal appendages, including hair, teeth, and nails, begin as a thickening of the ectoderm, called a placode. The placode arises from a primary induction signal that is sent from the underlying mesenchyme to the overlying epidermis. In mammals, the precise arrangement of hair follicles in the skin is due to the amount and distribution of signals that promote and inhibit hair placode formation. Continued development of a hair follicle after placode formation requires a complex cross-talk between the mesenchyme and epidermis. Here, I will review recent studies in humans and mice that have increased our understanding of the role of these signaling pathways in normal development and in hereditary hair loss syndromes. The study of normal hair development may suggest ways to restore or eliminate hair and might identify possible targets for the therapy of basal cell carcinoma, a cancer which strongly resembles embryonic hair follicles.     

Fibroblast growth factor receptor 2 (Fgfr2) plays an important role in eyelid and skin formation and patterning.

Initiating as protruding ridges above and below the optic vesicle, the eyelids of mice grow across the eye and temporarily fuse in fetal life. Mutations of a number of genes disrupt this developmental process and result in a birth defect, "open-eyelids at birth." Here we show that a critical event for eyelid induction occurs at embryonic day 11.5 (E11.5) when the single cell-layered ectoderm in the presumptive eyelid territory increases proliferation and undergoes morphologic transition to form cube-shaped epithelial cells. Using embryos lacking the Fgfr2 Ig domain III (Fgfr2(DeltaIII/DeltaIII)) generated by tetraploid rescue and chimeric embryo formation approaches, we demonstrate that this event is controlled by Fgfr2 signals as the Fgfr2(DeltaIII/DeltaIII) mutation blocks these changes and results in embryos without eyelids. Fgfr2 and its ligands are differentially expressed in the ectoderm and underlying mesenchyme and function in a reciprocal interacting loop that specifies eyelid development. We also demonstrate that similar defects account for failure of skin formation at early stages. Interestingly, Fgfr2-independent skin formation occurs at E14.5 mutant embryos, resulting in much thinner, yet well-differentiated epidermis. Notably, mutant skin remains thin with decreased hair density after transplantation to wild-type recipients. These data demonstrate an essential role of Fgfr2 in eyelid and skin formation and patterning.     

Origin of the vertebrate inner ear: evolution and induction of the otic placode

The vertebrate inner ear forms a highly complex sensory structure responsible for the detection of sound and balance. Some new aspects on the evolutionary and developmental origin of the inner ear are summarised here. Recent molecular data have challenged the longstanding view that special sense organs such as the inner ear have evolved with the appearance of vertebrates. In addition, it has remained unclear whether the ear originally arose through a modification of the amphibian mechanosensory lateral line system or whether both evolved independently. A comparison of the developmental mechanisms giving rise to both sensory systems in different species should help to clarify some of these controversies. During embryonic development, the inner ear arises from a simple epithelium adjacent to the hindbrain, the otic placode, that is specified through inductive interactions with surrounding tissues. This review summarises the embryological evidence showing that the induction of the otic placode is a multistep process which requires sequential interaction of different tissues with the future otic ectoderm and the recent progress that has been made to identify some of the molecular players involved. Finally, the hypothesis is discussed that induction of all sensory placodes initially shares a common molecular pathway, which may have been responsible to generate an 'ancestral placode' during evolution.     

Origin of the vertebrate inner ear: evolution and induction of the otic placode

The vertebrate inner ear forms a highly complex sensory structure responsible for the detection of sound and balance. Some new aspects on the evolutionary and developmental origin of the inner ear are summarised here. Recent molecular data have challenged the longstanding view that special sense organs such as the inner ear have evolved with the appearance of vertebrates. In addition, it has remained unclear whether the ear originally arose through a modification of the amphibian mechanosensory lateral line system or whether both evolved independently. A comparison of the developmental mechanisms giving rise to both sensory systems in different species should help to clarify some of these controversies. During embryonic development, the inner ear arises from a simple epithelium adjacent to the hindbrain, the otic placode, that is specified through inductive interactions with surrounding tissues. This review summarises the embryological evidence showing that the induction of the otic placode is a multistep process which requires sequential interaction of different tissues with the future otic ectoderm and the recent progress that has been made to identify some of the molecular players involved. Finally, the hypothesis is discussed that induction of all sensory placodes initially shares a common molecular pathway, which may have been responsible to generate an 'ancestral placode' during evolution.     

Apoptosis and proliferation in the trigeminal placode

The neurogenic trigeminal placode develops from the crescent-shaped panplacodal primordium which delineates the neural plate anteriorly. We show that, in Tupaia belangeri, the trigeminal placode is represented by a field of focal ectodermal thickenings which over time changes positions from as far rostral as the level of the forebrain to as far caudal as opposite rhombomere 3. Delamination proceeds rostrocaudally from the ectoderm adjacent to the rostral midbrain, and contributes neurons to the trigeminal ganglion as well as to the ciliary ganglion/oculomotor complex. Proliferative events are centered on the field prior to the peak of delamination. They are preceded, paralleled and, finally, outnumbered by apoptotic events which proceed rostrocaudally from non-delaminating to delaminating parts of the field. Apoptosis persists upon regression of the placode, thereby exhibiting a massive “wedge” of apoptotic cells which includes the postulated position of the “ventrolateral postoptic placode” (Lee et al. in Dev Biol 263:176–190, 2003), merges with groups of lens-associated apoptotic cells, and disappears upon lens detachment. In conjunction with earlier work (Washausen et al. in Dev Biol 278:86–102, 2005) our findings suggest that apoptosis contributes repeatedly to the disintegration of the panplacodal primordium, to the elimination of subsets of premigratory placodal neuroblasts, and to the regression of placodes.     

我国三种常见蛙类的预定水晶体分化对眼泡的依赖性及其眼泡诱导水晶体的能力

本文采用切除和移植实验,研究了黑斑蛙、泽蛙和狭口蛙预定水晶体的分化,对眼泡的依赖性及其眼泡诱导水晶体的能力。实验结果表明,脱离眼泡以后,3种蛙的预定水晶体均不能进一步分化,属于“依赖分化型”。至于眼泡诱导水晶体的能力大小,在3种蛙中有明显差别:黑斑蛙的眼泡不仅能诱导头部普通表皮,而且也可诱导腹部表皮形成水晶体;泽蛙眼泡对腹部表皮的诱导能力则较弱;而狭口蛙的眼泡仅能诱导头部普通表皮形成水晶体。本文讨论了造成这种差别的原因,认为除了眼泡的诱导能力之外,外胚层本身的反应能力及其时相的差异,也是应予考虑的因素。还讨论了温度可能影响眼杯分化以及水晶体的大小影响眼杯的大小等问题。此外,还报道了1例预定水晶体接触前脑顶壁,发生次生眼杯的事实。     

The role of growth factors in the embryogenesis and differentiation of the eye

The vertebrate eye is composed of a variety of tissues that, embryonically, have their derivation from surface ectoderm, neural ectoderm, neural crest, and mesodermal mesenchyme. During development, these different types of cells are subjected to complex processes of induction and suppressive interactions that bring about their final differentiation and arrangement in the fully formed eye. With the changing concept of ocular development, we present a new perspective on the control of morphogenesis at the cellular and molecular levels by growth factors that include fibroblast growth factors, epidermal growth factor, nerve growth factor, plateletderived growth factor, transforming growth factors, mesodermal growth factors, transferrin, tumor necrosis factor, neuronotrophic factors, angiogenic factors, and antiangiogenic factors. Growth factors, especially transforming growth factor-β, have a crucial role in directing the migration and developmental patterns of the cranial neural-crest cells that contribute extensively to the structures of the eye. Some growth factors also exert an effect on the developing ocular tissues by influencing the synthesis and degradation of the extracellular matrix. The mRNAs for the growth factors that are involved in the earliest aspects of the growth and differentiation of the fertilized egg are supplied from maternal sources until embryonic tissues are able to synthesize them. Subsequently, the developing eye tissues are exposed to both endogenous and exogenous growth factors that are derived from nonocular tissues as well as from embryonic fluids and the systemic circulation. The early interaction between the surface head ectoderm and the underlying chordamesoderm confers a lens-forming bias on the ectoderm; later, the optic vesicle elicits the final phase of determination and enhances differentiation by the lens. After the blood–ocular barrier is established, the internal milieu of the eye is controlled by the interactions among the intraocular tissues; only those growth factors that selectively cross the barrier or that are synthesized by the ocular tissues can influence further development and differentiation of the cells. An understanding of the tissue interactions that are regulated by growth factors could clarify the precise mechanism of normal and abnormal ocular development.     

The electrical properties of the ectoderm in the amphibian embryo during induction and early development of the nervous system

1. The electrical properties of ectodermal cells have been studied in embryos of the axolotl Ambystoma mexicanum between gastrulation and the closure of the neural tube.2. At the time of neural induction by the underlying mesoderm the mean membrane potential recorded in ectoderm cells was -30 mV (+/- 1.5 mV S.E. of mean) and in presumptive neural cells -27 mV (+/- 1.6 mV S.E. of mean).3. At late neural fold stages, when specification of the neuroectoderm is complete, the membrane potential in presumptive nerve cells was -44 mV (+/- 1.7 mV S.E. of mean). This is significantly greater than in cells of the surrounding ectoderm at the same developmental stage (-31 mV +/- 1.5 mV S.E. of mean).4. Current injected into an ectoderm cell spread freely throughout the neural and lateral ectoderm both before and after neural specification was complete.5. Voltage-current relations recorded at mid-neural fold stages in the lateral ectoderm and neural plate rectified in opposite directions. In the neural plate the slope conductance rose as the internal potential was made less negative; in the lateral ectoderm the slope conductance fell with depolarization.6. At the time of closure of the neural tube ectoderm and presumptive neural cells lose their low resistance connexions with each other. At the same time low resistance contacts are established across the mid line between ectoderm cells originally separated by the neural plate.7. After the neural tube has closed low resistance connexions remain between presumptive neural cells, although the degree of current spread from one cell to the next is not very great.8. The voltage-current relation recorded in neural tube cells showed a rise in slope conductance as the cell was depolarized.9. Occasionally signs of regenerative activity were seen, but the mechanism for generating a fully fledged action potential does not differentiate until after complete closure of the neural tube.