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Development of the Limbs

Biology 208. Developmental Biology.Carlson, 4th ed. Chapter 10.

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Web Lecture from Temple University

• http://isc.temple.edu/marino/embryology/limbs97/ppframe.htm

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Limb Bud Formation

• The distal tip of the limb bud is covered with a transient structure known as the apical ectodermal ridge (AER). Limb outgrowth is dependent upon interactions between the AER and the underlying mesoderm.

• If the AER is removed, the mesoderm stops dividing.

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Limb Development

• If a supernumerary AER is grafted adjacent to a developing limb, a supernumerary limb results.

• Prospective limb mesoderm grafted under flank ectoderm will promote formation of an AER and a supernumerary limb.

• If limb bud mesoderm is removed from an early limb bud, the AER regresses and the mesoderm ceases proliferation.

• Therefore, limb outgrowth involves reciprocal interactions between the AER and underlying mesoderm.

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Axes of Development

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What Controls Establishment of Limb Axes?

• The limb has three axes: Proximo-distal; Anterior-posterior and Dorsal-ventral.

• Proximo-distal

Removal of the AER at progressively later stages results in a truncated limb with progressively more distal elements. Thus, these elements are laid down in a proximal-to-distal direction.

The AER does not regulate P-D polarity. Fates of cells are determined by the length of time they spend in the progress zone. Those residing there the longest become the most distal elements.

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Anterior-Posterior Axis

This axis is determined by the zone of polarizing activity (ZPA), which is located at the junction between the limb bud and the body wall.

Grafts of the ZPA to the anterior margin of a host limb bud causes duplication of digits in mirror-image symmetry.

A diffusible morphogen (retinoic acid?) is released from the ZPA.

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Which axis forms first?

• 1. anterior/posterior (1st to 5th digits)

• 2. ventral/dorsal (palm/ dorsum of hand)

• 3. proximal/distal (base of limb / digits)

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Turtle skeleton

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Ambystoma maculatum

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Tuatara

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Tuataras

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Limbs of the chicken

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Mammalian Limbs

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Mammalian Limbs

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Primate Limbs

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Primate Limbs: Gorilla

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Human Anatomy

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How has the orientation of the limbs changed?

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Differentiation and Determination of the Limb Bud

• Myogenic regulatory factors (MRFs) are required for their differentiation

• Differentiation of myogenic cells in culture is arrested by growth factors, such as FGF and TGF-ß

• These results have led to the hypothesis that differentiation of myogenic precursors occurs in response to low growth factor levels (Konigsberg, 1971; Yaffe, 1971).

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Limb Development: Hox Genes

• Homeobox (Hox) Genes: Master Developmental Genes

• 1st identified in Drosophila• Regulate development of body segments • Homologous genes are present from

Arthropods to Vertebrates, highly conserved• Crucial to development, but functions are

poorly understood

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Human HOX Genes

• 39 different Hox genes distributed in 4 linkage groups

• Homeobox domain is highly conserved• Homeobox domain encodes a small protein

called homeodomain protein segment• This protein binds DNA and regulates gene

activity (i.e., it is a transcription factor)• Hox genes are expressed in sequences that

correlate with development of specific regions

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Homeobox Regulation of Gene Expression

From Human Development, by Professor Danton O’Day.

www.utm.utoronto.ca/%7Ew3bio380/lecture20.htm

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Hox Gene Expression in the Mammalian Limb

• The HoxD gene complex is expressed in a specific pattern in the developing mouse forelimb. The pattern of gene expression correlates with the linear arrangement of the genes in the genome.

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This figure shows a summary of the expression of the Hox genes relative to the final developed limb

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Hox-9 expressed earliest; Hox-13 expressed only in final stages of

digit formation

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Retinoic Acid : A Developmental Morphogen

• Retinoic acid is the active form of Vitamin A

• Both excess retinoic acid and vitamin A deficiency each affect left-right symmetry (e.g., abnormal formation of the mammalian heart) through alterations in gene expression.

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Cell Death and Digit Formation

Without apoptosis, the fingers would be webbed.: syndactyly

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• www.ucidevcell.org/faculty/susan-bryant/– For information on salamander limb development

and regeneration

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Gallus gallus (Chick) Limb Development-Retinoic Acid and Sonic Hedgehog (SHH)

Normal chick wing

A bead soaked in Retinoic Acid was implanted into the anterior margin of the early wing limb-bud. Note the mirror-image duplication of the digits.IVIIIII___

IIIIIIV

Digit IV represents a posterior limb structure. The ectopic release of Retinoic Acid from the bead leads to ectopic

expression of Sonic Hedgehog (SHH), forming a secondary ZPA.

www.luc.edu/faculty/wwasser/dev/chicklmb.htm

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• www.acs.ucalgary.ca/~browder/limb_dev.html

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