Molecular & Developmental Biology (BIOL3530)

With Dr. Brian E. Staveley
Department of Biology
Memorial University of Newfoundland

Development of Nematodes, Sea Urchins, Ascidians, and Slime Molds

Mosaic versus regulative development
Mosaic development depends upon localized cytoplasmic factors while regulative development depends upon cell-cell interactions.
In some invertebrates, cell fate is often specified at the single cell level, not in groups of cells, and does not rely upon positional information.
This process results from asymmetric cell division to distribute the cytoplasm unequally and allow determination of different cell fates.
Examination of developmental processes in distantly related species allows comparison.

Model Organism:  Caenorhabditis elegans

Small nematodes that are 1 mm long and 70 um in diameter.
The adult hermaphrodite (some males) undergo rapid development.
The egg has a 50 um diameter which forms a polar body after fertilization, nuclear fusion occurs followed by a set pattern of cleavage.
The normal pattern of cell division has been mapped.
Many cells undergo programmed cell death.
The first larval stage (L1) arises at 20 hours after fertilization.

Technique: Gene Silencing by RNA interference and microRNAs

Reversible gene silencing techniques rely upon the introduction of RNA complementary to the specific mRNA studied.
1) Short synthetic antisense RNA's (such as the chemically modified morpholino RNAs) bind to and inhibit the expression of mRNA.
2) RNA interference (RNAi) recruits the naturally occuring RNA-degrading mechanism to destroy the activity of a selected endogenous gene.
The enzyme Dicer chops dsRNA into 21-23 lengths which unwind into single stranded short interfering RNA (siRNA).
The siRNA incorporate into the RNA-induced silencing complex (RISC) riboprotein complex.
The nuclease component of RISC (Argonaute or Slicer) regrade mRNA based upon the exact complementarity of the siRNA.

Gene silencing by microRNAs is a natural mechanism to prevent the translation of mRNA.
The primary transcript can fold to generate hairpin RNA's that are cleaved by Dicer and are incorporated as guides into complexes.
Unlike siRNAs, the miRNAs are typically not perfect matches (one ore more mismatched basepairs).
Once bound the complex render the mRNA inactive to suppress translation.
These may not be degraded although many plant miRNA's are exact matches and undergo degration like siRNA.
Although a recently discovered aspect of biology, approximately 1% of human genes may encode miRNAs.

The complete genome of C. elegans has been sequences to reveal 20,000 genes.
Approximately 1700 genes affect development, many reveal via RNAi.

Cell lineage of Caenorhabditis elegans

The complete cell lineage of Caenorhabditis elegans is invariant.
The larva of 558 cells grows to an adult of 959 cells (plus germ cells).  131 undergo programmed cell death.
No polarity exist in the unfertilized egg but sperm entry controls the first cleavage.
A cap of actin microfilaments form at the anterior end and P granules become localized to the posterior.
The AB cell becomes the anterior and P is posterior.
P granule movement is the result (not a cause) of posterior determination.
maternal par-1 involved in establishing posterior identity.

Cell lineage of Caenorhabditis elegans
Axes depend on asymmetric division & cell interactions.
The 1st division gives a large anterior AB cell and a small P1 cell.  (P1, a stem cell, produces a P* cell and another.)
During first three divisions, P cells give rise to a number of lineages but the P4 then gives rise to only germ cells.
The AB cell divides into anterior ABa (neurons, epidermis plus pharynx mesoderm) and the posterior ABp (neurons, epidermis and specialized cells).
P1 divides into P2 and EMS which divides to make MS (mesodermal pharynx) and E (gut).
P2 becomes P3 and C (epidermis & muscle).
P3 becomes P4 and D (muscle).

Caenorhabditis elegans: cell-cell interactions
Cell-cell interactions specify cell fate in the early nematode embryo.
Cell fate is invariant but experiments reveal that cell-cell interactions are crucial.
ABp must contact the P2 cell (or it becomes an ABa cell).
glp-1 encodes a transmembrane receptor, uniform mRNA but translation is repressed in the P cell and is restricted to the AB cell.
After 2nd cleavage, P2 expresses apx-1 protein on its surface which activates Glp-1 receptor which causes ABa and ABp descendants to respond to signals from the MS cell differently.
Cell fate is directly linked to the pattern of cell division.
The fate map can be made at the 80-cell gastrula stage.

Caenorhabditis elegans: homeobox genes
A small cluster of homeobox genes specify cell fate along the A/P axis.
Four homeobox genes similar to the HOM/HOX genes plus an additional less related homeobox gene, make up the C. elegans Hox cluster.
They are expressed in different positions along the A/P axis.
Expressed during embryogenesis, their primary role is in larval development.

Caenorhabditis elegans:  heterochrony
Temporal information is important in the nematode.
Heterochronic mutants alter the timing of developmental events.
lin-14 mutants affect the T.ap (epidermis, neurons and support cells).
These mutants lead to precocious or retarded development.
gain-of-function (gf) mutations result in retarded development (happens later than normal).
loss-of-function (lf) mutations result in precocious development (happens earlier than normal).

Induction during Caenorhabditis elegans vulva development

The generation of the C. elegans vulva provides a model of developmental regulation.
The initial 3 responding cells give rise to 22 cells that divide, move and fuse in a precise pattern to generate 7 rings.
The anchor cell produces a signal (LIN-3) that induces a primary fate and a secondary fate through interaction with the LET-23 receptor.
The cell that adopts the primary fate, inhibits adjacent cells via lateral inhibition (via LIN-12).

Cell lineage of Echinoderms (sea urchins & starfish)

The sea urchin fate map is well defined but experiments have revealed a great deal of regulation is possible in this system.
The oral-aboral axis is usually at 45 degree angle to the first division.
Isolation of cells at the four cell stage results in  4 small sea urchins.
During the 4th cleavage, 4 small animal cells divide equally but the 4 large vegetal cells undergo asymmetric division to generate 4 vegetal micromeres and 4 macromeres which multiply into 1000 ciliated cells enclosing the blastocoel.
Gastrulation starts with entry of the primary mesenchyme (mesoderm) which lay down skeletal rods, followed by the endoderm and secondary mesenchyme which together stretch across the blastocoel to form the digestive tract.
Maternal factors specify the animal/vegetal axis and the vegetal organizer in the micromeres through a complex developmental network.
Important developmental genes have been shown to possess complex and modular regulatory pattern.
Sea urchins have a large capacity to regulate along the A/V axis with signals similar to that of the frog D/V axis.

Cell lineage of Ascidians

Ascidian larvaeappear to be similar to vertebrate neurulas (notochord, neural tube and muscles).
The egg and early embryo are regulative but after a few cleavages, the cells are mostly determined.
A fate map can be produced by the 110-cell stage.
The myoplasm (yellow pigmented in Styela embryos) give rise to the muscles of the larval tail by apparent mosaic development.
However, the notochord is induced by vegetal cells and involves the homologue of the Brachyury (T) gene (Regulative development).

Cell lineage of  the Cellular Slime Mold (Dictyostelium discoideum)

Cellular slime molds are eukaryotes that diverged earlier than plant and animals and share properties with each (with animals: cell movements during morphogenesis; with plants: cellulose cell walls).
"Dicty" is unicellular but aggregates to form a multicellular "slug" (~100,000 cells) during famine.
The slug forms a fruiting body with a mass of spore cells on top of a stalk.
In the slug, the pre-spore cells are located at the posterior and the pre-stalk cells are at the anterior.
When the slug rests, the pre-stalk cells migrate through the pre spore cells to form the stalk to push the pre-spore cells up into the air.
This is a very promising developmental system to study.

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