Developmental Specification &

Differentiation
Differentiation is the generation of specialized cell types (or we could say that: a cell is
said to be differentiated when it can perform its specific function). Before differentiation
can happen, the cell commits to a certain fate (i.e. commitment of the cell to a certain
fate).

The process of commitment can be divided into two stages:

1. The first stage is called specification. The fate of a cell or a tissue is said to be
specified when it is capable of differentiating autonomously (i.e., by itself) when
placed into a Petri dish or test tube. At the stage of specification, cell commitment is
still capable of being reversed.
2. The second stage of commitment is determination. A cell or tissue is said to be
determined when it is capable of differentiating autonomously even when placed into
another region of the embryo.

There are three major types of specification: (1) Autonomous Specification, (2)
Conditional Specification, and (3) Syncytial Specification. Embryos don’t use just one
mode of specification; rather multiple types of specification are used.

Autonomous Specification
In autonomous specification, the cell’s fate is determined by particular morphogenetic
determinants present in its cytoplasm (that it inherited from the egg); its fate is not
affected by surrounding cells.

The blastomere inherits a set of transcription factors from the egg cytoplasm, and these
transcription factors regulate gene expression, directing the cell into a particular path of
development. In other words, the egg cytoplasm contains different morphogenetic

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determinants (transcription factors or their mRNAs), which will influence the cell's
development.

Figure: Autonomous (mosaic) specification. (A-C) Differentiation of trochoblast (ciliated)

cells of the mollusk Patella. (D-G) Differentiation of a Patella trochoblast cell
isolated from the 16-cell stage and cultured in vitro. Even in isolated culture, these
cells divide and become ciliated at the correct time.

For instance, in tunicate (phylum Urochordata) embryos, each blastomere will form
most of its respective cell types even when separated from the remainder of the
embryo. In the 8-cell embryo, the two blastomeres that are going to generate tail
muscles contain a yellow-pigmented cytoplasm that has a muscle-specific transcription
factor called Macho. This transcription factor comes from the egg cytoplasm, and any
blastomere that has this factor will become muscle cells, even if that blastomere is
isolated from the rest of the embryo. If Macho containing cytoplasm is placed into other

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cells, those cells will form tail muscles. If the cells normally containing this cytoplasm
are removed from the embryo, the embryo will not form tail muscles. Hence, the tail
muscles of tunicates are formed autonomously by inheriting a transcription factor from
the egg cytoplasm.

Figure: Autonomous specification in the early tunicate embryo. When the four
blastomere pairs of the 8-cell embryo are separated, each forms the structures it would
have formed had it remained in the embryo.

Mosaic Embryos: Mosaic embryos are the embryos that get specified via autonomous
specification, since they develop like a mosaic of individually laid tiles — with each
cell receiving its instructions independently, without cell-cell interactions.

Embryos don’t rely only on Autonomous Specification: Even if most of the cells of an
early embryo are determined by autonomous specification, it does not imply that the
entire specification happens this way. Even in tunicate embryos, the nervous system
arises conditionally by cell interactions (conditional specification).

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Conditional Specification
Conditional specification is the ability of cells to achieve their respective fates by
interactions with other cells. In this case, what a cell becomes is largely determined by
paracrine factors secreted by its neighbors. Many scientists such as August Weismann
and Wilhelm Roux tried to disapprove conditional specification (they believed that
specification was only autonomous) but it was approved eventually thanks to the work
of Hans Driesch.

Figure: Conditional specification. (A) What a cell becomes depends on its position in
the embryo. Its fate is determined by interactions with neighboring cells. (B) If cells
are removed from the embryo, the remaining cells can regulate and compensate
for the missing part.

Work of August Weismann: In 1888, August Weismann proposed the germ plasm
theory, in which each cell of the embryo would develop autonomously. He proposed that
the sperm and egg provided equal chromosomal contributions to the new organism.
Moreover, he postulated that the chromosomes carried the inherited potentials of this
new organism. However, not all the determinants on the chromosomes were thought to
enter every cell of the embryo. Instead of dividing equally, the chromosomes were
hypothesized to divide in such a way that different chromosomal determinants entered

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different cells. The fertilized egg would carry all of the determinants, but certain somatic
cells would retain the "blood-forming" determinants while others retained the "muscle-
forming" determinants, and so forth. Only the nuclei in those cells which were to
become germ cells (gametes) were postulated to contain all the different types of
determinants. The nuclei of all other cells would have only a subset of the original
determinants.

Figure: Weismann's germ plasm theory of inheritance. The germ cell (blue) gives rise
to the differentiating somatic cells of the body, as well as to new germ cells.
Weismann hypothesized that only the germ cells contained all the inherited
determinants. The somatic cells were each thought to contain a subset of the
determinants, and the types of determinants found in a somatic cell's nucleus would
determine its differentiated type.

Weismann also proposed a hypothesis based on the fate map of the frog embryo, that
when the first cleavage division separated the future right half of the embryo from the
future left half, there would be a separation of "right" determinants from "left"
determinants in the resulting blastomeres.

Work of Wilhelm Roux: Wilhelm Roux tested Weismann’s hypothesis by using a hot
needle to kill one of the cells in a 2-cell frog embryo—with the result that only half of the

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larva developed. Based on this result, Roux claimed that specification was mosaic
(autonomous specification) and that all the instructions for normal development were
already inside each cell. (Note: August Weismann and Wilhelm Roux tried to reject the
idea of conditional specification, they believed that the only mechanism of specification
was autonomous/mosaic).

Figure: Roux's attempt to demonstrate autonomous specification. Destroying (but not

removing) one cell of a 2-cell frog embryo resulted in the development of only one half
of the embryo.

In reality, Roux's experiments had a defect that was that they answered the question of
how the remaining blastomeres of an embryo would develop when a subset of
blastomeres was destroyed, but they did not prove Weismann's hypothesis.

Work of Hans Driesch (Evidence for Conditional Specification): Hans Driesch (1892)
obtained opposite results as compared to Roux. Driesch performed isolation
experiments. He separated sea urchin (phylum Echinodermata) blastomeres from each
other by vigorous shaking (or by placing them in calcium-free seawater). Surprisingly,
each of the blastomeres from a 2-cell embryo developed into a complete larva.
Similarly, when Driesch separated the blastomeres of 4- and 8-cell embryos, some of
the isolated cells produced entire pluteus larvae (larva of sea urchins).

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Figure: Driesch's demonstration of conditional (regulative) specification. (A) An intact
4- cell sea urchin embryo generates a normal pluteus larva. (B) When the 4-cell embryo
is removed from its fertilization envelope and each of the four cells are isolated, each
cell can form a smaller, but normal, pluteus larva.

This result was very different from the predictions of Weismann and Roux. Rather than
self-differentiating into its future embryonic part, each isolated blastomere regulated its
development to produce a complete organism. These experiments provided the first
experimentally observable evidence of conditional specification.

In conditional specification, the fate of cells depends on the cells' neighbors. Interactions
between cells determine their fates, rather than some cytoplasmic factor that is
particular to that type of cell.

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Driesch confirmed conditional development in sea urchin embryos by performing a
recombination experiment. In sea urchin eggs, the first two cleavage planes are
normally longitudinal (vertical/meridional), whereas the third division is equatorial
(horizontal), dividing the embryo into four upper and four lower cells.

Figure: Normal cleavage in 8- to 16-cell sea urchin embryos, seen from the animal pole
(upper sequence) and the side (lower sequence). The nuclei are numbered.

Driesch (1893) changed the direction of the third cleavage by gently compressing early
embryos between two glass plates, thus causing the third division to be meridional
(vertical/longitudinal) like the first two. After he released the pressure, the fourth division
was equatorial (horizontal).

This procedure reshuffled the nuclei, placing nuclei that normally would have been in
the region destined to form endoderm into the presumptive ectoderm region. In other
words, some nuclei that would normally have produced ventral structures were now
found in the dorsal cells.

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Figure: Cleavage under pressure. Abnormal cleavage planes formed under pressure,
as seen from the animal pole and the side. Some nuclei (such as 6A and 8A) are placed
in different regions of the embryo.

If segregation of nuclear determinates had occurred (as had been proposed by

Weismann and Roux), the resulting embryo should have been strangely disordered
(because the cells were displaced). However, Driesch obtained normal larvae from
these embryos. He thus concluded that:

“The relative position of a blastomere within the whole will probably in a general way
determine what shall come from it.”

Results of Driesh’s Experiments: The results of Driesch’s experiments proved

conditional specification:

1. First, Driesch had demonstrated that the prospective potency of an isolated

blastomere (i.e., the cell types that are possible for it to form) is greater than the

blastomere's prospective fate (those cell types it would normally give rise to over the
unaltered course of its development). It means that if a blastomere is separated from
the embryo, it will not follow its previous fate (when it was a part of the embryo) but it
will instead create a new embryo (due to its prospective potency). According to

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 Weismann and Roux, the prospective potency and the prospective fate of a
blastomere should have been equal/same.

2. Second, Driesch concluded that the sea urchin embryo is a "harmonious

equipotential system" because all of its potentially independent parts interacted
together to form a single organism. Driesch's experiment implies that cell interaction
is critical for normal development. Moreover, if each early blastomere can form all
the embryonic cells when isolated, then it means that, in normal development, the
community of cells must prevent it from doing so.

3. Third, Driesch concluded that the fate of a cell depended only on its location in the
embryo. The interactions between cells determined their fates.

Regulative Embryos: Regulative embryos are the embryos (especially vertebrates) in

which most of the early blastomeres are conditionally specified.

Sea Urchin embryos use both Autonomous and Conditional Specification: In the 16-
cell sea urchin embryo, a group of cells called the micromeres inherit a set of
transcription factors from the egg cytoplasm. These transcription factors cause the
micromeres to develop autonomously into the larval skeleton. These transcription
factors also activate the genes for paracrine factors and juxtacrine factors that are
employed by the micromeres to conditionally specify the cells around them (both
paracrine and juxtacrine factors are chemical messengers that affect neighboring cells,
juxtacrine factors require physical contact between cells, paracrine factors don’t require
it). Similarly, the frog embryo also uses both autonomous and conditional specification.

Syncytial Specification
A cytoplasm that contains many nuclei is called a syncytium, and the specification of
presumptive cells within such a common cytoplasm is called syncytial specification. In

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early embryos of insects, nuclei divide within the egg; but the cell does not divide. In
other words, many nuclei are formed within one common cytoplasm.

The insect egg cytoplasm is not uniform. Nuclei in the anterior part of the cell will be
exposed to cytoplasmic transcription factors that are not present in the posterior part of
the cell, and vice versa. The interactions of nuclei and transcription factors take place in
a common cytoplasm.

Each nucleus in Drosophila is given positional information (i.e., whether that nucleus is
to become part of the anterior, posterior, or midsection of the body) by transcription
factors acting as morphogens. These transcription factors are made in specific sites in
the embryo, diffuse over long distances, and form concentration gradients where the
highest concentration is at the point of synthesis and gets lower as the morphogen
diffuses away from its source. The concentration of specific morphogens at any
particular site tells the nuclei where they are in relation to the source of the
morphogens.

Morphogen Gradients & Cell Specification: A morphogen (Greek, "form-giver") is a

diffusible biochemical molecule that can determine the fate of a cell by its concentration.

Morphogens can be transcription factors produced within cells. They can also be
paracrine factors that are produced in one group of cells and then travel to another
population of cells, specifying the target cells differentially according to the
concentration of morphogen.

Uncommitted cells exposed to high concentrations of the morphogen (nearest its source
of production) are specified as one cell type; when the morphogen's concentration drops
below a certain threshold, the cells are determined to another fate. When the
concentration falls even lower, a cell of the same initial uncommitted type is specified in
yet a third manner.

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Morphogens (involved in syncytial specification) differ from morphogenetic determinants
because they quantitatively specify cells, while a morphogenetic determinant (which is
involved in autonomous specification) specifics cells in a qualitative manner.

Morphogens involved in the Syncytial Specification of Drosophila Embryo: The

anterior-most portion (front) of the Drosophila embryo produces a morphogen called
Bicoid with a concentration that is highest in the anterior and declines toward the
posterior (anterior to posterior gradient).

The posteriormost portion of the egg forms a posterior to anterior (back to front)
gradient of the morphogen Caudal (and Nanos). Thus, the longitudinal axis of the
Drosophila egg has opposing morphogen gradients—Bicoid coming from the anterior,
and Caudal (and Nanos) from the posterior.

Bicoid and Caudal are both transcription factors, and different concentrations and ratios
of Bicoid and Caudal (and Nanos) proteins activate different sets of genes in the
syncytial nuclei.

 Formation of head in Drosophila Embryo: Those nuclei in regions containing high

amounts of Bicoid and little Caudal (and Nanos) are instructed to activate those
genes necessary for producing the head.

 Formation of thorax in Drosophila Embryo: Nuclei in regions with slightly less

Bicoid and a small amount of Caudal (and Nanos) are instructed to activate
genes that generate the thorax.

 Formation of abdomen in Drosophila Embryo: Nuclei in regions that have little or

no Bicoid but plenty of Caudal (and Nanos) are instructed to form the abdominal
structures.

Syncytial Specification is followed by Autonomous/Conditional Specification: Thus

when the syncytial nuclei are eventually incorporated into cells, these cells will have

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their general fate specified. Afterward, the specific fate of each cell will become
determined both autonomously (from the transcription factors acquired by the cell's
nucleus from the egg cytoplasm) and conditionally (by interactions between the cell and
its neighbors).

Figure: Syncytial specification in Drosophila melanogaster. Anterior-posterior

specification originates from morphogen gradients in the egg cytoplasm, specifically the
morphogenetic transcription factors Bicoid and Caudal. The concentration of Bicoids
is higher in the anterior region (anterior to posterior gradient) whereas the concentration
of Caudal (and Nanos) is higher in the posterior (posterior to anterior gradient).
These varying morphogen gradients activate different genes in nuclei in different
regions. After cellularization, the cells from different body parts of the fly.

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