Thelimb bud is a structure formed early invertebratelimb development. As a result of interactions between theectoderm and underlyingmesoderm, formation occurs roughly around the fourth week of development.^[1] Inthe development of the human embryo the upper limb bud appears in the third week and the lower limb bud appears four days later.^[2]

Limb bud
Details
Precursor	lateral plate mesoderm
Identifiers
Latin	gemmae membrorum
MeSH	D018878
TE	bud_by_E5.0.3.0.0.0.5 E5.0.3.0.0.0.5
Anatomical terminology [edit on Wikidata]

The limb bud consists of undifferentiated mesoderm cells that are sheathed in ectoderm.^[3] As a result of cell signaling interactions between the ectoderm and underlying mesoderm cells, formation of the developing limb bud occurs asmesenchymal cells from thelateral plate mesoderm andsomites begin to proliferate to the point where they create a bulge under the ectodermal cells above.^[4] The mesoderm cells in the limb bud that come from the lateral plate mesoderm will eventually differentiate into the developing limb's connective tissues, such ascartilage,bone, andtendon.^[3] Moreover, the mesoderm cells that come from thesomites will eventually differentiate into themyogenic cells of the limbmuscles.^[3]

The limb bud remains active throughout much of limb development as it stimulates the creation andpositive feedback retention of two signaling regions: theapical ectodermal ridge (AER) and thezone of polarizing activity (ZPA) with the mesenchymal cells.^[3] These signaling centers are crucial to the proper formation of a limb that is correctly oriented with its corresponding axial polarity in the developing organism. Research has determined that the AER signaling region within the limb bud determines the proximal-distal axis formation of the limb usingFGF signals.^[5]ZPA signaling establishes the anterior-posterior axis formation of the limb usingShh signals.^[6] Additionally, though not known as a specific signaling region like AER andZPA, the dorsal-ventral axis is established in the limb bud by the competitiveWnt7a andBMP signals that the dorsal ectoderm and ventral ectoderm use respectively.^[7][8] Because all of these signaling systems reciprocally sustain each other's activity, limb development is essentially autonomous after these signaling regions have been established.^[3]

TheHox genes, which define features along the anterior-posterior axis of a developing organism, determine at which points along the axis that limb buds will form.^[9] Though limbs emerge at different locations in different species, their positions always correlate with the level of Hox gene expression along the anterior-posterior axis.^[9] All limb buds must also rely on other signaling factors to obtain their forelimb or hindlimb identity; Hoxgene expression influences expression ofT-box proteins that, in turn, determine limb identity for certain organisms.^[3]

In turn, the activation of T-box protein activates signaling cascades that involve theWnt signaling pathway andFGF signals.^[3] Before limb development begins, T-box proteins initiateFGF10 expression in theproliferating mesenchymal cells of the lateral plate mesoderm, which form the limb bud mesoderm.^[3]WNT2B andWNT8C stabilize thisFGF10 expression in the forelimb and hindlimb, respectively.^[10][11] ThisFGF10 expression stimulatesWNT3 expression in the above ectodermal cells – resulting in formation of the apical ectodermal ridge as well as inducingFGF8 expression.^[12] TheFGF8 secreted by the AER acts to keep the cells of the limbmesenchyme in a mitotically active state and sustains their production ofFGF10.^[12]positive feedback loop between the limb mesenchymal cells and the AER maintains the continued growth and development of the entire limb.^[13]

In addition to limb outgrowth, the formation of a crucial signaling center, thezone of polarizing activity (ZPA), in a small posterior portion of the limb bud helps to establish anterior-posterior polarity in the limb through secretion of the proteinSonic hedgehog (Shh).^[3] The ZPA also plays an important role in initially specifying digit identity, while later maintaining proper AER morphology and continuedFGF8 secretion – to ensure proper mitotic activity of the limb bud mesenchyme beneath.^[3]

In chickens,Tbx4 specifies hindlimb status, whileTbx5 specifies forelimb status.^[13] In mice, however, both hindlimbs and forelimbs can develop in the presence of eitherTbx4 orTbx5.^[14] In fact, it is thePitx1 andPitx2 genes that appears to be necessary for specification of the developing hindlimb, whereas their absence results in forelimb development.^[15]Tbx4 andTbx5 appear to be important specifically for limb outgrowth in mice.^[14]

Within the limb bud, expression of specificHox genes varies as a function of the position along the anterior-posterior axis. TheHox genes are linked in four chromosomal clusters: Hoxa, Hoxb, Hoxc, and Hoxd.^[9] Their physical position on thechromosome correlates with the time and place of expression. This statement is supported by the knowledge thatHox gene expression is initiated duringgastrulation inprimitive somitic mesoderm by FGF signaling which effects the primitive somitic mesoderm cells at different times depending on their axial location during organismdevelopment—and is even further specified with other anterior-posterior axis signals (such asretinoic acid).^[3] Additional evidence for the role thatHox genes play inlimb development was found when researchers effectedHox gene expressions inzebrafish by addingretinoic acid duringgastrulation; This experiment resulted in a duplication of limbs.^[16] Although excess retinoic acid can alter limb patterning by ectopically activating Shh expression, genetic studies in mouse that eliminate retinoic acid synthesis have shown that RA is not required for limb patterning.^[17]

Chicken development is a wonderful example of this specificity ofHox gene expression in regard to limb development. The most 3’ Hoxc genes (HOXC4,HOXC5) are expressed only in the anterior limbs in chickens, while the more 5’ genes (HOXC9,HOXC10,HOXC11) are expressed only in the posterior limbs.^[9] The intermediate genes (HOXC6,HOXC8) are expressed in both the upper and lower limbs in chickens.^[9]

As previously stated, limb development is essentially autonomous after the signaling centers (AER) andZPA) have been established. However, it is important to know thatHox genes continue to participate in the dynamic regulation of limb development even after the AER andZPA have been established in the limb bud. Complex communication ensues as AER-secretedFGF signals andZPA-secretedShh signals initiate and regulate Hox gene expression in the developing limb bud.^[18] Though many of the finer details remain to be resolved, a number of significant connections between Hox gene expression and the impact on limb development have been discovered.

The pattern ofHox gene expression can be divided up into three phases throughout limb bud development, which corresponds to three key boundaries in proximal-distallimb development. The transition from the first phase to the second phase is marked by the introduction ofShh signals from theZPA.^[19] The transition into the third phase is then marked by changes in how the limb bud mesenchymal cells responds toShh signals.^[19] This means that althoughShh signaling is required, its effects change over time as the mesoderm is primed to respond to it differently.^[19] These three phases of regulation reveal a mechanism by whichnatural selection can independently modify each of the three limb segments – thestylopod, thezeugopod, and theautopod.^[19]

FGF10 can induce limb formation, but T-box proteins, Pitx1, and Hox genes determine identity^[1]

By mimicking the initial FGF10 secretions of the lateral plate mesoderm cells,limb development can be initiated. Other signaling molecules are implicated in determining the limb's identity.

1. Placement of FGF10-containing beads beneath chick ectodermal cells results in the formation a limb bud, AER,ZPA and, subsequently, an entire limb. When the beads created limb buds towards the anterior region, forelimb formation coincided withTbx5 expression, while hindlimb formation coincided withTbx4 expression. When beads were placed in the middle of the flank tissue, the anterior portion expressed Tbx5 and forelimb features, while the posterior portion of the limb expressed Tbx4 and hindlimb features.
2. When chick embryos were engineered to constitutively expressTbx4 (via viral-transfection) throughout their flank tissue, every limb they grew was a leg, even those that formed in the anterior region, which would normally become wings. This confirms the role ofT-box proteins in the type of limb that develops.
3. Knocking outTbx4 orTbx5 knockout preventsFGF10 expression in the lateral plate mesoderm in mice.
4. TheHox pathway affects Tbx expression, which in turn affectsFGF10 expression.^[3]
5. WhenPitx1 was incorrectly expressed in mouse forelimbs, several hindlimb-associated genes (Tbx4,HOXC10) were turned on and drastic alterations of the muscles, bones, and tendons shifted the phenotype towards that of a hindlimb. This indicates thatPitx1—throughTbx4—plays a role in the emergence of hindlimb properties.

HOXD11 expression correlates with Shh signals secretion^[20]

HOXD11 is expressed posteriorly, near the ZPA, where the highest levels ofShh signal expression occur.

1. Whenretinoic acid is applied to induceShh signal expression, aZPA is transplanted, or ectopic expression ofShh signaling is stimulated, HOXD11 expression follows.

Mesenchymal cells determine limb identity, but the AER maintains limb outgrowth throughFGF signal secretion^[1]

These experiments reveal that the limb mesenchyme contains the necessary information concerning limb identity, but the AER is needed to stimulate the mesenchyme to live up to its destiny (of becoming an arm, leg, etc.)

1. When the AER is removed, limb development halts. If anFGF-bead is added in the AER's place, normal limb development proceeds.
2. When an extra AER is added, two limbs form.
3. When forelimb mesenchyme is replaced with hindlimb mesenchyme, a hindlimb grows.
4. When forelimb mesenchyme is replaced with non-limb mesenchyme, the AER regresses, and limb development halts.

ZPA's role in establishing polarity and further limb development^[21]

TheZPA first specifies anterior-posterior polarity (and dictates digit identity), and then, by sustaining AER activity, it ensures that the necessary cell proliferation occurs for normal formation of a five-digit limb.

1. When Shh signals normally secreted from theZPA are inhibited (either through use oftamoxifen orShh-null mutants) the AER morphology, particularly its anterior extent, is perturbed and itsFGF8 signaling decreased. As a result ofShh downregulation during limb bud expansion, the number of digits was decreased, but the identities of the formed digits was not altered.

Associated molecules include:^[1]

• FGF10 – Initially, Tbx proteins induce secretion of FGF10 by cells in the lateral plate mesoderm. Later,FGF10 expression is restricted to the developing limbmesenchyme, where it is stabilized by WNT8C orWNT2B.FGF10 expression activates secretion ofWNT3A, which acts upon the AER and inducesFGF8 expression. The mesenchyme, throughFGF10 secretion, is involved in a positive feedback loop with the AER, throughFGF8 secretion.
• FGF8 – Secreted by the AER cells. Acts upon the mesenchymal cells, to maintain their proliferative state. Also induces the mesenchymal cells to secreteFGF10, which acts throughWNT3A to sustain the AER's expression ofFGF8.
• WNT3A – Acts as a middle man in thepositive feedback loop between the AER and limb mesenchyme. Activated byFGF10 expression, activatesFGF8 expression.
• Sonic hedgehog^[20] Secreted by the ZPA in the limb bud mesenchyme. Creates concentration gradient that dictates formation of the five distinct digits. Digit 5 (pinkie) results from exposure to highShh concentrations, while digit 1 (thumb) on the opposite end of the spectrum develops in response to low concentrations ofShh.Shh expression has been shown in many, but not all circumstances, to be heavily connected withHox gene expression.Shh also (viaGremlin) blocksbone morphogenic protein (BMP) activity. By blockingBMP activity, FGF expression in the AER is maintained.
• Tbx4,Tbx5 – Involved with the development of hindlimbs versus forelimbs. Though in chicks, they seem to be the primary factors involved in limb identity, in mice it appears thatTbx4 is merely a downstream messenger enforcing the hindlimb-forming instructions ofPitx1. WhetherPitx1 merely diverts a prospective forelimb from that path to become a hindlimb, or ifTbx5 is activated by another Pitx1-like messenger, is unknown.
• Pitx1 – Responsible for the development of a hindlimb-associated phenotype.Tbx4 is one of its downstream targets. Pitx1 and Tbx4 encodetranscription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induced distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 was misexpressed developed into limbs with some morphological characteristics of hindlimbs.^[22]
• Hox genes – Responsible for dictating the anterior-posterior axis of an organism, and are intricately involved in patterning of the developing limb in conjunction withShh. Influences the activity ofT-box proteins and FGF signals (and possiblyPitx1) proteins. Determines where limb buds will form, and what limbs will develop there.