Limb Development Flashcards
Development of the limbs
LIMB PRIMORDIA
PROCESSES INVOLVED IN LIMB DEVELOPMENT
DIFFERENTIATION
SPECIFICATION OF LIMBS
HOX GENES IN LIMB DEVELOPMENT
LIMB ABNORMALITIES
LIMB PRIMORDIA
LATERAL PLATE MESODERM
BONES
TENDONS AND LIGAMENTS
VASCULATURE
SOMITE MESODERM
LIMB MUSCLES
NCC
DERMIS
MELANOCYTES
SCHWANN CELLS
NEURAL TUBE
NEURONS
ORGANOGENESIS - DEVELOMENT OF ORGANS - MAJOR EVENT OF EMBRYONIC AND FETAL PERIODS
WILL LOOK AT DEVELOMENT OF THE LIMB AS ANEXAMPLE OF ORGANOGENSIS
THIS SLIDE SHOWS PRIMORDIA THAT GIVE RISE TO THE LIMBS
Morphogenesis-deelopment of form
NCC= Neural Crest Cells
Schematic of myoblast migration from ventrolateral dermomyotome into limb buds
Figure 3 (A) Schematic of myoblast migration from ventrolateral dermomyotome into limb buds. (B) Immunostaining of transverse section of HH stage 20 chicken embryo. PAX3 (red) -positive cells are shown delaminating and entering limb bud (red arrowheads). (C) Schematic of dorsal and ventral muscle masses formed by limb bud myoblasts. Undifferentiated precursors (some of which will give rise to satellite cells in adult muscles) are shown in red with differentiated myotubes shown in green. (D) Immunostaining of transverse section of HH stage 24 chicken embryo. PAX3 (red) cells are restricted to the dorsal and ventral regions of the limb bud. My, myotome; Dm, dermomyotome; P, proximal; D, distal; dmm, dorsal muscle mass; vmm, ventral muscle mass; AER, apical ectodermal ridge of limb bud.
myotome cells blue migrating into limb bud
DEVELOPMENTAL PROCESSES INVOLVED IN LIMB MAKING
MORPHOGENESIS
MITOSIS
CELL MIGRATION
CELL RECOGNITION
CELL ADHESION
DIFFERENTIATION*
INDUCTION*
CELL SIGNALING*
APOPTOSIS
PATTERN FORMATION*
AXIS FORMATION*
MOLECULES INVOLVED:
GROWTH FACTORS
MORPHOGENS
TRANSCRIPTION FACTORS
MITOSIS - NEEDED FOR GROWTH OF LIMB
CELL MIGRATION - SOMITE MESODERM MIRGATES INTO LIMB AND BECOMES MUSCLE
CELL RECOGN AND ADHESION/ IMPORTANT IN HISTOGENESIS - FORMATION OF TISSUES WITHIN THE LIMB
DIFFERENTIATION - DEVELOPMENT OF MESENCHYME INTO TISSUES SUCH AS MUSCLE OR BONE
INDUCTION - INFLUENCE OF ONE TISSUE ON DEVELOPMENT OF ANOTHER
CELL SIGNALING - INVOLVED IN INDUCTIONS
APOPTOSIS - CELL DEATH - IN LIMB - DESTROYS WEBBING BETWEEN DIGITS
DETERMINATION AND DIFFERENTIATION
GENERATION OF CELL DIVERSITY
DUE TO DIFFERENTIAL GENE EXPRESSION
HOUSEKEEPING GENES ARE ON IN ALL CELLS
CERTAIN GENES GET TURNED ON IN MUSCLE TO GIVE IT THE CHARACTERISTICS OF MUSCLE
ACTIN AND MYOSIN - LUXURY PROTEINS EXPRESSED IN MUSCLE
IF BONE WERE PRODUCED, A DIFFERENT SET OF GENES WOULD BE TURNED ON
determination: transcription factor(s) dictate to (mesenchymal) cells that it will become (muscle), even though it looks the same, it’s determined to be something else
Differentiation-when cells can actually be determined to be different
GENES INVOLVED IN MAKING A SKELETAL MUSCLE–MyoD GENE FAMILY
Myf5
MyoD
Myogenin
MRF4
PERTAINS TO SKELETAL MUSCLE
ONE OF BEST UNDERSTOOD SYSTEMS FOR DETERMINATION- DIFFERENTIATION
IS REGULATED BY THE MYOD GENE FAMILY - GROUP OF MYOGENIC REGULATAORY FACTORS (MRF)
TRANS FACTORS THAT TURN ON MUSCLE LINEAGE DURING DEV
THE MYOD GENES ENCODE HELIX LOOP HELIX (HLH) TYPE TRANSCRIPTION FACTORS
BIND TO CANNTG SEQUENCE IN PROMOTERS OR ENHANCERS OF OTHER GENES AND TURN THEM ON
MASTER SWITCHES -
MYOD GENES - TWO FUNCTIONS:
COMMITS UNDETERMINED CELL TO MUSCLE LINEAGE
ACTIVATES GENE THAT PRODUCE MUSCLE SPECIFIC PROTEINS
WHERE DOES THIS HAPPEN?
IN SOMITES – SPECIFICALLY IN THE MYOTOME OF SOMITES
MYF5 IS EXPRESSED IN THE DORSAL-MEDIAL SOMITE – LATER BECOMES MUSCLE OF BACK (EPAXIAL)
MYOD IS EXPRESSED IN DORSALATERAL SOMITE – LATER BECOMES MUSCLE OF BODY WALL AND LIMB
BOTH MYF5 AND MYOD ARE TURNED ON BY WNT PROTEINS (SIGNALING) FROM DORSAL NEURAL TUBE AND SURFACE EPIDERMIS RESPECTIVELY
MyoD encodes transcription factors
1st transcription factors during myogenesis (Muscle formation): Myf5 or MyoD
KO MyoD–> mice still had Myf5, made muscle
KO Myogenin–>no muscle
In situ hybridisation of MYF5 in HH stage 16 embryos
Figure 2 (A) In situ hybridisation of MYF5 in HH stage 16 embryos. Anterior is towards the top, posterior is towards the bottom. Expression is seen in anterior somites starting in the epaxial myotome and then spreading more widely through the somite. Posterior somites (I–VI) do not express muscle-specific genes. (B) In situ hybridisation of MYF5 in HH stage 22 embryos. Expression is seen in all muscle types including myogenic cells in somites (s), limb buds (lb) and head. Tongue muscle precursors are seen in the hypoglossal cord (hgc), jaw and facial muscles in the branchial arches (ba) and extraocular muscles (eom) around the eye.
Comparison of the signalling pathways leading to MRF expression and myogenesis in epaxial and hypaxial somite, limb, head and neck, and adult
Figure 4 Comparison of the signalling pathways leading to MRF expression and myogenesis in epaxial and hypaxial somite, limb, head and neck, and adult. In grey boxes are the core network of MRFs in activating myogenesis from different tissues. Positive regulatory signals are shown in red, and inhibitory signals are shown in blue. Black arrows show genetic interactions. (A) Epaxial somite: DACH2, SIX1/4, EYA2, PAX3, MYF5 and MRF4 induce MYOD, thus leading to myogenin (MYOG) expression and myogenesis. WNT1, WNT3A and SHH up-regulate MYF5 expression, and WNT7A up-regulates MYOD. BMP4 negatively regulates MYOD but is itself inhibited by noggin. (B) Hypaxial somite: DACH2, SIX1/4 and EYA2 are capable of inducing PAX3. Subsequently, PAX3 induces MYF5 and MRF4, which in turn activate MYOD expression, followed by MYOG expression and myogenesis. BMP and WNT7A positively regulate MYF5 and MYOD expression. (C) Limb: DACH2, SIX1/4, EYA2, PAX3, MEOX2 and PITX2 are capable of inducing MYF5 and MYOD; subsequently, MYOD induces MYOG expression and myogenesis. MSX1 can inhibit MYOD expression. BMP and HGF up-regulate PAX3 expression, and WNT regulates MSX1. (D) Head and neck: PITX2 and TBX1 induce each other and MYF5. TBX1 is capable of inducing MYF5 expression (and MRF4 in those head muscles which express it) before activating MYOD expression. Subsequently, MYOD induces MYOG expression followed by myogenesis, but inhibitory signals from WNT and BMP can also regulate MYOG expression. (E) Adult: PAX3/7 directly induce MYF5 expression, whereas SIX1/4 can induce MRF4, MYF5 and MYOG. MRF4 and MYOD activate MYOG expression followed by myogenesis.
TRANSFECTION OF NON-MUSCLE CELL WITH MyoD
REVIEW EXPERIMENT
SHOWS MyoD IS SUFFICIENT TO CAUSE MUSCLE CELL DIFFERENTIATION
KNOCKOUTS
KO MYOD IN MICE
EXPECT - NO MUSCLE
GET MUSCLE OK
OTHER GENES IN MYOD FAMILY CAN SUBSTITUTE FOR MYOD EG. MYF-5 CAN SUB FOR MYOD
KO MYOGENIN - DEVELPOMENT STOPS - NO BACKUP
IN LIMB, MUSCLE DEVELOPS FROM SOMITE MESENCHYME THAT
MIGRATES INTO LIMB BUD
MESENCHYME FROM LP FORMS CARTILAGE, BONES, BLOOD, CT
INDUCTION
HANS SPEMMAN AND HILDE MANGOLD
1924
PRIMARY INDUCTION = INDUCTION OF NEURAL TUBE FORMATION BY PS AND NOTOCHORD
SPEMMAN AND MANGOLD DISCOVERED
DRAW ON BOARD EXPT SHOWING DISCOVERY
MANY OTHER INDUCTIONS TAKE PLACE DURING DEVELOPMENT - THESE = SECONDARY INDUCTIONS
SECONDARY INDUCTIONS MAY BE RECIPROCAL
Image is recriprocal induction
INDUCTION – WAYS IT MAY OCCUR
LIMB BUDS (INDUCTION)
INDUCTION DURING LIMB DEVELOPMENT
EXPTS SHOWING RECIPROCAL INDUCTION BETW LIMB MESENCHYME AND AER
- NO LIMB
CONCLUDE : MESENCHYME IS NEC FOR LIMB FORMATION - LIMB MESENCHYME AT ECTOPIC SITE INDUCES FORMATION OF AER AND A NEW LIMB FORMS
CONCLUDE: MESEN HAS INFO TO DIRECT LIMB FORMATION; CAN INDUCE FORMATION OF AER AND CAN INSTRUCT AER AT ECTOPIC SITE - WHEN HINDLIMB MESEN AND FORELIMB MESEN ARE COMBINED A HIND LIMB FORMS
CONCLUDE: MESEN CLOSEST TO AER CONTROLS WHAT DEVELOPS
EVIDENCE FOR INTERACTION OF MESEN W/ ECTODERM
growing limb bud has only mesenchyme, surrounded by a layer of ectoderm which thickens at the tip–>apical ecodermal ridge
INDUCTION DURING LIMB DEVELOPMENT AER EXPERIMENTS
EXPTS WITH ECTODERM
- NO NEW LIMB FORMS IF AER GRAFTED TO NEW SITE
CONCLUDE: ECTODERM ALONE WILL NOT PROMOTE LIMB DEVELOPMENT - IF AER REMOVED, LIMB DEVELOPMENT STOPS
CONCLUDE: AER IS ALSO NEC FOR LIMB DEVELOPMENT
AER INFLUENCES MESENCHYME
RECIPROCAL INDUCTION - EXTRA STRUCTURE FORMS
CONCLUDE: ECTODERM EXERTS INFLUENCE ON MESENCHYME
RECIPROCAL INDUCTION
OVERALL CONCLUSIONS:
A. MESENCHYME INDUCES FORMATION OF AER AND INSTRUCTS AER TO PRODUCE A CERTAIN TYPE OF LIMB
B. BUT AER IS NEC FOR SUSTAINED LIMB DEVELOPMENT AND GROWTH
REGIONAL SPECIFICITY OF INDUCTION
DERMIS OF SKIN COMES FROM MESODERM
EPIDERMIS OF SKIN COMES FROM ECTODERM
RECOMBINATION EXPTS:
- MESODERM DETERMINES WHICH CUTANEOUS STRUCTURE FORMS
- ECTODERM DEVELOPS ACCORDING TO THE REGION FROM WHICH THE MESODERM WAS TAKEN
- MESENCHYME IS INSTRUCTIVE CAUSES DIFFERENT GENES TO BE TURNED ON IN RESPONDING CELLS
GENETIC SPECIFICITY OF INDUCTION
THE MESENCHYME CAN INSTRUCT THE ECTODERM AND TELL IT WHAT SET OF GENES TO TURN ON BUT THE MESENCHYME CAN COMPLY ONLY WITH WHAT IS IN IT GENOME!
GENETIC SPECIFICITY OF INDUCTION CONTINUED
CHICK BRANCHIAL ARCHES HAVE NOT MADE TEETH IN 10 MILLION YEARS
CHICK ECTODERM STILL RETAINS GENE FOR TOOTH MAKING
CHICK MUST HAVE LOST MOLECULES THAT INDUCE EXPRESSION OF TOOTH MAKING GENES DURING EVOLUTION - SO THEY NO LONGER MAKE TEETH
THE MOUSE MESENCHYME SUPPLIED THE INDUCER SO TEETH WERE MADE
INTERESTING INSIGHT INTO HOW EVOLUTION OCCURS
NOT NECESSARY TO LOSE GENE TO LOSE A STRUCTURE
HERE A STRUCTURE WAS LOST BEC/ THE INDUCER WAS LOST!!
