Gallus gallus (Chick) – Limb Development
Egg is large, extremely yolky, telolecithal. Cytoplasm located in small island on top of yolk mass. Embryonic development can be observed by cutting window through eggshell.
Cleavage: meroblastic. Cell divisions do not penetrate yolk mass.
Blastula: epiblast formed on top of yolk mass, as bilaminate (two-layered) disk of cells. Cells of upper layer will form mesoderm and ectoderm, cells of lower layer will form endoderm.
Gastrulation: cells of upper layer migrate into space between cell layers through Hensen’s node, at anterior end of epiblast, and the primitive streak which extends posteriorly from Hensen’s node.
Neurulation: late in gastrulation, cells anterior to Hensen’s node flatten out into neural plate, lateral ridges grow upwards and fuse to form neural tube.
Organogenesis: internal organs, limbs and bone form.
Left-right asymmetry (heart loops to right) accompanied by transient expression of signalling molecules on only one side of embryo:
– activin receptor (ActRII) on right side of primitive streak shortly after start of gastrulation
– sonic hedgehog (Shh) and nodal expressed at 1 hr of gastrulation on left side of node
– nodal at 5 hrs of gastrulation on left lateral plate mesoderm
– Snail-related (cSnR) expressed in right lateral plate mesoderm, and suppression of cSnR expression randomizes heart looping, without affecting nodal
Figure 1. Developmental Anatomy of the Chick Wing (Johnson & Tabin, Cell, Vol. 90, 979-990, September 19, 1997, Copyright � 1997 by Cell Press)
(A) Schematic of a chick embryo at about 50 hr of incubation. The presumptive forelimbs region is located within the lateral plate mesoderm adjacent to somites 16-20. At this stage, the presumptive hindlimb region resides adjacent to paraxial mesoderm that has not yet segmented.
(B) Schematic of a chick embryo at about 72 hr of incubation. At this stage the presumptive limbs appear as buds jutting out from the flank.
(C) Schematic of a chick wing at 10 days. By this stage, the basic adult pattern of the wing has been realized as a cartilaginous model: a single long bone, the humerus, is present most proximally, followed by two long bones, the radius and ulna. At the distal end are the wrist (carpal) and digit (phalanges) elements. The three cardinal axes of the limb are indicated to the right of the schematic. For comparisons between forelimbs and hindlimbs of different species, it is often useful to refer to the homologous regions as stylopod (upper limb), zeugopod (middle limb), and autopod (distal limb).
(D) Scanning electron micrograph (SEM) section of a 50 hr chick embryo. The limbs form from the lateral plate mesoderm, and a migratory contribution from adjacent somites. The intermediate mesoderm lies in between the lateral and somitic mesoderm.
(E) SEM section of the forelimb of a 72 hr chick wing bud. At this stage, limb buds have a relatively simple histological profile with a mesenchymal core surrounded by an ectodermal jacket. The apical ectodermal ridge (AER) is located at the distal tip of the bud. Micrographs in (D) and (E) courtesy of Gary C. Schoenwolf, University of Utah School of Medicine.
Limb buds form on either side of embryonic axis, from lateral mesoderm.
Dissected limb bud treated with trypsin (a protease) leaves intact ectoderm.
Dissected limb bud treated with EDTA (chelator of divalent metal ions) leaves intact mesoderm.
Graft of limb bud mesoderm into ectopic site induces ectopic limb bud formation.
Graft of limb bud ectoderm has no effect.
What regulates patterning and growth of the limb bud?
Apical Ectodermal Ridge (AER) regulates proximo-distal patterning:
Limb bud mesoderm induces formation of ectodermal ridge at distal end, called the apical ectodermal ridge (AER).
Removal of AER stops further outgrowth of limb. Distal parts fail to form.
Graft of AER to another limb bud induces secondary outgrowth from point of graft. Recipient limb bud, with 2 AERs, thus develops two sets of distal structures.
Maintenance of AER depends on factor from mesoderm. Mesoderm from wingless mutant + wild type ectoderm with AER causes regression of AER, no limb.
AER maintenance factor appears to be concentrated in posterior zone of mesoderm.
Zone of Polarizing Activity (ZPA) regulates anterior-posterior polarity:
Rotation of entire tip of wing bud & regrafting to stump yields mirror-image duplicatiaon of distal parts.
Posterior of tip remains determined, produces posterior digits. Anterior of tip, now relocated to posterior, changes fate and also produces posterior digits, with mirror image and opposite handedness.
Diffusible posterior determinant in stump mesoderm realigns A-P polarity in duplicated digits, but original dorso-ventral polarity maintained, thus produces opposite symmetry.
Grafting of tip to after 180 degree rotation to intact wing bud can produce 4 sets of digits. Original posterior regions of host and graft produce posterior digits; anterior regions of both reorient to produce mirror-image duplications.
Posterior determinant located in posterior mesoderm: Zone of Polarizing Activity (ZPA).
ZPA grafted onto apex of bud induces secondary digits in normal orientation; e.g., 2-3-4- 3-4.
ZPA grafted onto anteior of bud induces mirror image duplication: e.g., 4-3-2-3-4.
ZPA grafted onto posterior has no effect.
Role of non-AER ectoderm in Limb Development/ Patterning
It has been well established that AER plays a major role in limb development of chicken embryo as it promotes proliferation and survival of the underlying limb mesoderm. The survival of the limb mesoderm is important since limb buds are formed through interactions between mesoderm and ectoderm. A tudy done by Marian Fernandez-Teran, Maria A. Ros and Francesca V. Mariani explored the role of non-AER ectoderm in limb development by examining the relationship between the ectoderm and the mesoderm (Link to the paper). Non-AER has a role in limb development as it provides cell signals for dorssal and ventral patterning. The study was able to successfully demonstrate that non-AER ectoderm is required for the survival of adjacent mesoderm. Removal of the ectoderm resulted in damage to the mesoderm and in malformed limb skeletons and muscles (Link to the web page)
Dorsal Ectoderm regulates dorso-ventral polarity:
Signals from the ectoderm control D-V positioning of distal limb structures.
Reversal of limb bud ectoderm in grafts onto limb bud mesoderm causes reversal of distal structures.
Removal of dorsal ectoderm, more than ventral ectoderm, decreases ZPA activity.
ZPA produces diffusible morphogen whose concentration determines A-P position.
AER determines P-D position, possibly by # of cell divisions in progress zone (PZ).
Progress Zone located beneath AER, at A/P boundary. Cells leaving progress zone fixed in their development.
Stage 24 tip grafted onto stage 19 stump produces short limb with parts between 19 and 24 missing.
Stage 19 tip grafted onto stage 24 stump produces longer limb with parts between 19 and 24 duplicated.
Figure 3. The Progress Zone Model and Progressive Proximal-Distal Specification (Johnson & Tabin, Cell, Vol. 90, 979-990, September 19, 1997, Copyright � 1997 by Cell Press)
(Left) Progress zone (PZ) cells lie subjacent to the apical ectodermal ridge (AER, yellow). Under the influence of AER signals, the PZ cells acquire a P/D positional address (green).
(Middle) As cells within the progress zone proliferate, some of these cells leave the progress zone and are displaced proximally. Cells outside the influence of the progress zone retain their positional address when they exit the progress zone.
(Right) Cells that remain in the progress zone have their positional address adjusted to a more distal value (indicated by the orange color). Through repeated application of this mechanism, distal enlargement of the limb and P/D patterning could be coordinated.
What are the signalling molecules?
Retinoids have been known as teratogens.
Retinoic acid (RA) posteriorizes embryos.
Retinoic acid mimicz ZPA activity in limb buds:
Implants of beads coated with RA produces mirror-image duplications like ZPA grafts.
RA is present in limb buds at 25 nM concentration; 20 nM RA applied exogenously is sufficient to produce digit duplications.
Concentration of RA is higher at posterior: up to 50 nM.
Mammals have a plethora of receptors for retinoic acid: RAR-alpha, RAR-beta, and RAR-gamma, each with splice variants, and RXR (retinoid X receptor), as well as a cellular retinoic acid binding protein (CRABP).
RARs and RXR all belong to the type II nuclear hormone receptor family: they function as RAR/RXR heterodimers and bind specifically to DNA at retinoic acid response elements (RAREs) (Niederreither & Dollé 2008).
In the absence of ligand (RA), RAR/RXR heterodimers bind transcriptional repressors. The ligand (retinoic acid) diffuses across plasma membrane into the cell and into the nucleus to bind to the RAR/RXR heterodimeric receptor.
The receptor:ligand complex binds trancriptional activator proteins to activate transcription of target genes regulated by RAREs.
Among RA targets are hox genes, which have RAREs in their upstream regulatory sequences.
But is RA really the morphogen produced by the ZPA, or does it induce formation of ZPA?
Implants of RA beads result in formation of ZPA, not around the bead, but distal to the bead, and only 16-24 hours after implantation.
RA beads induce expression of RARs, but ZPA implants do not.
RA does appear to be required for limb bud initiation, in zebrafish, chick and mouse embryos. Raldh (retinaldehyde dehydrogenase) mutants or chemical inhibition of RA synthesis prevents limb bud initiation. RA is produced in the somites where limb buds form. See review by Duboc and Logan, 2011.
Sonic Hedgehog (SHH)
Homologue of Drosophila hedgehog (hh) gene, expressed in imaginal discs.
Both HH and SHH are secreted proteins, autoproteolytically cleaved, with short- and long-range signalling capability.
Shh expressed in posterior mesoderm of limb bud, in region coinciding with ZPA, with same timing as ZPA activity.
Shh induced by ectopic RA, coincides with ZPA activity induced by RA.
Ectopic expression of shh in anterior of limb bud (mediated through chick fibroblasts infected with retroviral vector) results in mirror-image digit duplications.
SHH induces FGF-4 expression in AER.
SHH induces BMP2 (member of TGF superfamily; homolog of Drosophila decapentaplegic).
Ectopic SHH activates Hoxd genes in same manner as ZPA implants.
Shh knockout mice have multiple defects in neural tube patterning, axial patterning, and loss of distal limb structures (Chiang et al. 1996).
Fibroblast Growth Factors (FGFs)
Various members of FGF family (FGF-2, FGF-4, FGF-8) expressed in AER.
Can replace AER to direct outgrowth and patterning of limb.
FGF-4, in particular, expressed in posterior of AER.
FGF-8 expressed uniformly throughout AER.
FGF-4 expression increases in anterior AER in response to ZPA graft.
FGF-4 implant in proximal region induces SHH & ZPA activity.
FGF-4 and PZ cells induce ectopic limb
Transplants of latex beads soaked in FGF-1, FGF-2, or FGF-4 into lateral interlimb mesoderm can induce ectopic limb; FGF-2 induces most complete limbs with digits (Cohn et al. 1995). However, none of these FGFs are expressed in lateral mesoderm prior to limb bud initiation.
FGF-8 implants can also induce ectopic limb formation, and is expressed in lateral interlimb mesoderm at time of limb bud initiation (Crossley et al., 1996).
FGF10 knockout mice fail to form either forelimb or hindlimb buds (see review by Duboc and Logan, 2011).
Member of Wnt family of secreted signalling proteins.
Expressed in dorsal ectoderm of limb buds.
Drosophila wingless gene induces expression of hedgehog at D/V boundary
Wnt7a knockout mice show lack of dorsal structures on limbs (Parr & McMahon, 1995)..
Interactions among signalling molecules and axes
FGF-8 induces formation of limb bud ectoderm (AER), and expression of FGFs in AER.
FGF-4, FGF-8 from AER induces Shh in posterior mesoderm (ZPA) & regulates cell proliferation in progress zone.
SHH from ZPA induces FGF-4 in posterior of AER and polarizes limb mesoderm.
RA induces both Shh and FGF-4 – affects, D-V, A-P, and P-D axes.
Wnt7a from dorsal ectoderm is required for Shh expression and maintenance, establishes dorsal compartment. En establishes ventral compartment.
Boundary of D/V compartments, proximity to AER establish location of ZPA.
Cells in progress zone receive all 3 signals to establish pattern in developing limb bud.
Figure 4. Three Axes and Three Signals: Shh, FGFs, and Wnt-7a Orchestrate Limb Pattern (Johnson & Tabin, Cell, Vol. 90, 979-990, September 19, 1997, Copyright � 1997 by Cell Press)
(A) Schematic of a limb bud viewed from the posterior-dorsal aspect showing the localization of Shh to the ZPA, FGFs to the AER, and Wnt-7a to the dorsal ectoderm.
(B) Codependence of Shh, FGF, and Wnt-7a signaling and axial patterning. While each secreted factor can be associated with patterning along a single axis, affecting the expression of any single factor will lead to modulation of the other two. For example, reduction of Wnt-7a signaling will lead directly to dorsal patterning defects, but indirectly to posterior defects through a diminution of Shh signaling, and to proliferation defects via a subsequent effect on FGF expression.
Establishment of the AER at D/V boundary
Correct positioning of AER depends on boundary of expression of Radical Fringe (homolog of Drosophila fringe) (Rodriguez-Esteban et al., 1997; Laufer et al., 1997).
Radical Fringe expressed in dorsal ectoderm prior to formation of AER, and repressed in ventral ectoderm by Engrailed-1.
Misexpression of Radical Fringe or Engrailed-1 in ventral ectoderm disrupts AER or causes division of AER or ectopic AER.
Figure 2. Mechanisms of D/V Patterning and AER Positioning (Johnson & Tabin, Cell, Vol. 90, 979-990, September 19, 1997, Copyright � 1997 by Cell Press)
(A) Gene expression along the limb bud D/V axis. Wnt-7a and Radical fringe (r-Fng), which encode secreted factors, are expressed in the dorsal ectoderm. The homeodomain-containing factors encoded by Lmx-1 and Engrailed-1 (En-1) localize to the dorsal mesoderm and ventral ectoderm, respectively.
(B) Genetic interactions involved in AER formation and specification of dorsal pattern. En-1 expression in the ventral ectoderm restricts the expression of r-Fng and Wnt-7a to the dorsal ectoderm. Interaction between r-Fng-expressing and r-Fng-nonexpressing cells leads to the specification of the AER. Wnt-7a instructs the dorsal mesoderm to adopt dorsal characteristics, such as Lmx-1 expression, which in turn specifies dorsal pattern. En-1 has a dual function in AER positioning and dorsal specification and hence acts to coordinate the two processes.
Hox genes expressed in limb buds along proximal-distal axis, with 3′ gene expressed first (most proximally) and 5′ genes expressed last (most distally).
Targeted disruption of hoxd-11 and hoxa-11 simultaneously causes missing radius and ulna in mouse forelimbs (Davis et al., 1995).
Induction of ectopic limbs causes reprogramming of Hox (Hox b9, c9, d9) gene expression patterns in lateral plate mesoderm (Cohn et al., 1997).
Figure 5. Hox Genes and the Specification of Limb Bud Positional Information (Davis et al. 1995)
(A) Hox gene expression in the chick wing bud is quite dynamic, with several independently regulated phases of expression (shown here for Hoxd-10). In phase 1, Hox genes are expressed across the entire distal limb bud, during the time that the upper wing is specified. Subsequently in phase 2, Hox genes are expressed in a posteriorly nested order. A limb bud at the time the lower wing is specified shows overlapping expression in both phase 1 and 2 patterns. Finally, in phase 3 the Hox genes are expressed in a more distal pattern. At the time the digits are specified, the wing bud expresses the Hox genes in both phase 2 and phase 3 patterns.
(B) In the chicken limb bud, the relative order of expression of the Hox genes reverses between phase 2 and phase 3. Although the order of the HoxD genes is different in the zeugopod from that in the autopod, Sonic hedgehog (Shh) is able to induce these genes in the proper temporal and spatial order within each segment. Thus, the order in which Hox genes are activated in response to Shh is dependent upon the P/D segment of the limb bud on which Shh is acting. It is important to note that even though the Hox genes are centered around the Shh-expressing cells and can be activated by Shh, their expression is initiated in a posteriorly biased manner even in the absence of Shh (see text).
(C) Hox genes seem to function, in part, to drive the proliferation of the limb elements. There is a correspondence between the limb segments regulated by the Hox genes and both the order of the genes within the cluster and phase of Hox expression. For example, The lower wing is specified during phase 2, when Hoxa-11 and Hoxd-11 are broadly expressed. Due to “posterior prevalence” these genes have a greater role in this segment than more 3′ Hox genes; and the expression of more 5′ Hox genes, such as Hoxa-13 and Hoxd-13, is confined to the extreme posterior margin during this phase and hence does not have a major impact on the development of the lower wing. Thus, the double mutant lacking both Hoxd-11 and its paralog Hoxa-11 has an approximately normal upper limb and foot, but the lower limb exhibits little growth after the initial cartilage condensations form, and hence the lower limb segment is nearly missing.
Wing or Leg?
What determines whether a limb bud will produce a wing (forelimb) or leg (hindlimb)? Forelimb buds express a T-box transcription factor, TBX5, whereas hindlimb buds express TBX4 and Pitx1 (a paired-type homeobox transcription factor)(Science news article by Vogel, 1999). Misexpression of Pitx in the chick wing bud caused expression of TBX4 and the resulting limb looked more like a leg than a wing (Logan and Tabin, 1999).
TBX5 mutants do not form forelimb buds. TBX5 causes transcription of FGF10 in the mesenchyme, setting up a positive feedback loop for FGF10 to direct FGF8 expression in the AER, which in turn upregulates FGF10 expression in the distal mesenchyme.
In mice lacking TBX4, hindlimb buds do form, but do not grow. See review by Duboc and Logan, 2011, for discussion of roles of TBX5, TBX4, and PitX1 in determining limb identify and morphology. Ouimette et al. (2010) propose that forelimb is the default limb identity, and that TBX4 represses forelimb identity to enable hindlimb patterning by Pitx1 in mouse embryos.
Evolution of Limbs – a Developmental Genetics Perspective
Evolution of animal appendages – fins, wings, legs – can be modeled from the expression of common regulatory genes, and is consistent with evolution of the regulatory gene apparatus from a common ancestor (Shubin et al., 1997).
Gonadal and Embryonic Development – MHM gene
The chicken Z chromosome-linked locus MHM, is an integral part of a chicken’s normal embryonic and gonadal development. It is methylated and transcriptionally silent in male cells, but is hypomethylated and transcribed into a long non-coding RNA in female cells. It has been linked to sex-determination in the chicken embryo but lacks evidence. Variations of the MHM gene in both males and females can cause a plethora of abnormalities in the gonads and other parts of the chicken embryo’s anatomy2.The purpose of this research is to investigate the role of this gene using different variants of mis-expression of the sense and antisense strands (Roeszler et al., 2012).
Other Topics in Avian Development
- Recent advancement in the mechanisms involved in regulation of vascular differentiation in the yolk sac of the chick embryo (Link).
- Click here for more info on the neural mechanisms of language development in songbirds, specifically regarding the FOXP2 gene.
- Zebra finches are being used as a model organism to learn more about neural patterning in learning, memory, and sensorimotor integration – recently, the development of temporal structure was examined.
- Zebra finches have also been studied to identify the mechanistic pathways in which songbirds’ songs are processed and encoded. The roles of substructures within the brain and how they link together to encode the signals needed to reproduce their songs is discussed in the Roberts et al.’s paper Motor circuits are required to encode a sensory model for imitative learning.
- Canaries used a model organisms in order to understand the differences in sex and enhancements of testosterone in the telencephalic ventricle zone in the adult canary brain as discussed by Barker et al. (2013)
- Click here for a description about testosterone production, sexual dimorphic morphology, and digit ratio in dark-eyed juncos.
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|<strong>Role of non-AER ectoderm in Limb Development/Patterning</strong>||<strong>Role of non-AER ectoderm in Limb Development/Patterning</strong>|
|It has been well established AER plays a major role in limb development of chicken embryo as it promotes proliferation and survival of the underlying limb mesoderm. The survival of the limb mesoderm is important since limb buds are formed through interactions between mesoderm and ectoderm. A study done by <a href=”http://www.devbio.biology.gatech.edu/?page_id=11695″>Marian Fernandez-Teran, Maria A. Ros and Francesca V. Mariani</a> explored the role of non-AER ectoderm in limb development by examining the relationship between the ectoderm and the mesoderm. Non-AER has a role in limb development as it provides cell signals for dorsal and ventral patterning. The study was able to successfully demonstrate that non-AER ectoderm is required for the survival of adjacent mesoderm. Removal of the ectoderm resulted in damage to the mesoderm and malformed limb skeletons and muscles. (<a href=”http://www.devbio.biology.gatech.edu/?page_id=11695″>Link to the webpage</a>)|