3/24/2009Development of the Limbs Biology 208. Developmental Biology.Carlson, 4th ed. Chapter 10.

3/24/2009Web Lecture from Temple Universityhttp://isc.temple.edu/marino/embryology/limbs97/ppframe.htm

3/24/2009Limb 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.

3/24/2009Limb 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.

3/24/2009Axes of Development

3/24/2009What 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.

3/24/2009Anterior-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.

3/24/2009Which axis forms first?

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

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

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

3/24/2009Turtle skeleton

3/24/2009Ambystoma maculatum

3/24/2009Tuatara

3/24/2009Tuataras

3/24/2009Limbs of the chicken

3/24/2009Mammalian Limbs

3/24/2009Mammalian Limbs

3/24/2009Primate Limbs

3/24/2009Primate Limbs: Gorilla

3/24/2009Human Anatomy

3/24/2009How has the orientation of the limbs changed?

3/24/2009Differentiation 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-

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 DrosophilaRegulate development of body segments Homologous genes are present from Arthropods to Vertebrates, highly conservedCrucial to development, but functions are poorly understood

3/24/2009Human HOX Genes 39 different Hox genes distributed in 4 linkage groupsHomeobox domain is highly conservedHomeobox domain encodes a small protein called homeodomain protein segmentThis 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

3/24/2009

Homeobox Regulation of Gene Expression

From Human Development, by Professor Danton ODay.

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

3/24/2009Hox 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.

3/24/2009This figure shows a summary of the expression of the Hox genes relative to the final developed limb

3/24/2009Hox-9 expressed earliest; Hox-13 expressed only in final stages of digit formation

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3/24/2009Retinoic Acid : A Developmental MorphogenRetinoic 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.

3/24/2009Cell Death and Digit FormationWithout apoptosis, the fingers would be webbed.: syndactyly

3/24/2009www.ucidevcell.org/faculty/susan-bryant/For information on salamander limb development and regeneration

3/24/2009Gallus gallus (Chick) Limb Development-Retinoic Acid and Sonic Hedgehog (SHH)

Normal chick wingA 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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3/24/2009www.acs.ucalgary.ca/~browder/limb_dev.html

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