The Integument and Connective Tissues

Week 3 An introduction to microscopic anatomy

The objectives of the lab are:

To introduce you to basic aspects of histology, the study of tissues.

To study the integument at the gross and microscopic levels.

To examine bone and cartilage and gain an understanding of the major characteristics of these tissues.

To introduce you to the morphology of a vertebra (as opposed to vertebrate)

Diversity:

There is no diversity exercise, but instead you will have a chance to look at models of developmental stages of several different vertebrates.

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INTRODUCTION

In todays lab well do a number of different activities. You will see microscopes set up at each station. This should be your first activity to go through the microanatomy of the integument and then of bone and cartilage. Please read the appendix, which has a brief introduction to using the microscope. Also take time to set yourself up. Adjust your chair so you can comfortably see through the microscope and adjust the width between the eyes so that you can see through both oculars. You will see much more, and be more comfortable, if you take the time to do this.

Remember histology is the study of tissues, the anatomical level between cells and organs. Tissues provide the building blocks of the body. This will be the only lab in which histological study is central, but subsequent labs will have optional histological exercises. If you continue into any medical field you will spend considerable time understanding histology as it is important in pathology and clinical diagnosis. Please make sure you read the targeted reviews on Bone and Cartilage as well as on Histology for overviews of this material.

Par t I: The Integument

Recall from lecture that the integument or skin of vertebrates has a dual origin embryonically: the epidermis develops from epidermal ectoderm, the dermis from mesoderm. All vertebrates possess both an epidermis and a dermis. The dermis is lacking in cephalochordates and urochordates as well as other invertebrates.

The epidermis, as a tissue that is a covering sheet, is an example of an epithelial tissue. It may be simple (one cell layer) but is generally squamous (many cells thick). The integument also frequently contains glands which are also ectodermal in origin, even though they may lie in the dermal layer. Keratin, an extracellular matrix protein, is a product of the epidermis.

Compare sections of the skins of frogs (as representatives of semiaquatic animals) and terrestrial mammals. The dermis does is always composed of a variety of loose and dense connective tissues. However, in different sections of different animals and parts of the body you will see differences in the thickness of the keratinzed layer, as well as the epidermis and dermis.

Look first at slides of frog skin (see illustration on next page). The frog epidermis is thin and non-keratinized. Large glands, derived from the epidermis, project into the dermis. In favorable sections these will be seen to communicate with the surface via ducts. Most glands secrete mucous, which is important in keeping the frogs skin moist. In some species toxic substances are also secreted by specialized glands. Large, dark-staining cells between the epidermis and the glands are chromatophores: cells containing pigment which give the animal color. Many fish, amphibians, and reptiles have the ability to change color by expanding or contracting the pigment granules in the chromatophores. The dermis in frogs is fairly unspecialized.

Remember, there are 4 major types of tissue

1. Epithelial tissue (epithelium) is specialized for protection of surfaces, absorption, and secretion.

2. Connective tissue is specialized for joining other tissues together and it wraps and supports individual organs and the body as a whole. Connective tissue is characterized by large amounts of extracellular matrix.

3. Muscular tissue is specialized for contractility and conductivity. Well talk about the different types later in the course.

4. Nervous tissue is specialized for irritability and conductivity. In general, nervous tissue is highly receptive to stimuli, and when irritated, conducts waves of excitation over long distances.

Many sources define blood tissue (largely blood cells) as a 5th tissue type.

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Note: there are two different frog skin specimens, one is stained pinkish (as shown on the top). The other is stained with a different stain and looks blackish brown (the difference is due to preparation and not species differences). Make sure you look at both of them, and identify similar structures in each. The specimens stained blackish are of a species that has many more mucous

glands. The specimen has also been prepared such that a lot of the dermis has been removed. Look at both and compare.

Label the glands, pigment cells, epidermis and dermis in the slide to the left.

Next well look at several specimens of mammalian integument. The mammalian epidermis (and that of most amniotes) is much thicker and is generally covered with a layer of keratin. Its thickness, as well as the thickness of the keratin layer correlates primarily with the degree of abrasion that part of the skin receives. In general, the dermis is also much thicker in mammals. The specific structure of the dermis depends on the function of that part of the integument, as we discussed in class. Look at the following slides. Weve labeled some structure for you make sure you identify and label the following on each: epidermis, dermis, keratin. Label any glands, hairs or pigment cells you observe as well.

1) Human abdominal skin.

Identify keratin, epidermis and dermis in the human abdominal skin. Note the thickness of the dermal layer.

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2) Monkey skin

Examine the section of the monkey skin. Notice the hair cells, sebaceous glands and adipose tissue. Adipose tissue was also present under the human abdominal integument, it was removed before sectioning.

3) Integument of the foot

Compare the skin from the sole of the foot in a human and cat to the abdominal skin. Note the thickness of the epidermal layers and in particular the thick keratin layer. When you develop a callus, you are thickening this layer. Label the two sections below. The difference in color is due to staining technique.

Human foot Cat foot:

The staining in the cat renders the dead, keratinized epidermal cells bright yellow-green. Below this the brown layer consists of the germinative epidermal cells. The blue-green layer under the epidermis is the dermis. There are also copious fat deposits in the pads/soles of the foot.

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Now look at other structures such as bird feather (see illustration on the title page of a bird feather) and the human fingernail.

Now, take a break from the microscope and explore the various structures of the integument laid out for your inspection. Look at the quills of the African (big) and North American (smaller) porcupine. What structure are these homologous with? Note the armadillo armor. What layer is this derived from? Look at the baleen. Below weve shown a picture of how it fits in the whales mouth. Watch the video of the week on whale feeding. Examine other structures on display.

Photos of fingernail slide. Use key below to understand the anatomy. (Left fingernail tip; right fingernail base).

Whale baleen, used in filter feeding whales.

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Par t II Connective tissues car tilage and bone

Connective tissues are classified primarily on the basis of the relative amounts of the different structural components that are present. These components include cells of various types and also extracellular components.

A distinctive feature of connective tissue is the predominance of extracellular materials. There are two major categories of extra-cellular components: ground substance and fibers (which are produced by specialized cells called fibroblasts). Fibers include collagen fibers, which do not stretch and elastic fibers, composed of the protein elastin, which can stretch to 1.5 times resting length, as well as other fiber types.

Cartilage is a specialized form of connective tissue which has a firm and definite shape essential to its supporting function. It consists of a firm matrix of collagenous fibers surrounded by an amorphous matrix consisting of a protein-polysaccharide complex (proteoglycans). Chondrocytes (=cartilage cells) lie in lacunae (small cavities in the extracellular matrix). Cartilage is a major tissue in the skeletal system. There are three major types of cartilage, but today you will primarily look at hyaline cartilage, which is present in developing bone and also on many joint surfaces.

Bone is another specialized form of connective tissue and is the tissue which forms most of the adult skeletal system in the majority of vertebrates. The matrix of bone is very solid due to the deposition of calcium phosphate and other salts on the collagenous fibers. Bone tissue may have either of two textures: Spongy or cancellous bone consists of irregular trabeculae (beams) of bony tissue between which lie connective tissues and blood vessels. This is the first type of bone which forms during the development of dermal and cartilage replacement bones. Spongy bone is often reworked to form compact or lamellar bone. Compact bone consists of dense layers with few interspaces. Bone tissue of both of these textures may be formed either as endochondral ossification of as membrane bone.

Dermal or membrane bone has an intramembranous origin, i.e. its development begins with the secretion of collagen by strands of mesenchymal cells in connective tissue membranes. Osteoblasts (bone forming cells) differentiate and are responsible both for secreting the organic intercellular substance and directing the deposition of calcium salts into trabeculae.

Cartilage replacement or endochondral bone is structurally similar to dermal bone, but is formed by replacement of a cartilaginous model. The development of a skeletal element such as the humerus begins with the formation from mesenchyme of a cartilage precursor. Later in development the cartilage is invaded by periosteal tissue derived from the initial perichondrium. The cartilage model is eroded away and replaced by a bony element. During development the surviving fragments of cartilage serve as a framework for the deposition of bone tissue.

Make sure you review the glossary in the targeted review on bone and cartilage.

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We will look at a number of examples of bone and cartilage so that you can see the major differences between these tissues.

Look first at Slide I-50 which is an example of spongy bone, forming in a membrane. The calcium phosphate has been removed chemically leaving behind the collagenous fibers (stained red) and osteocytes (bone cells) trapped in lacunae. The bony tissue is seen as an open meshwork of trabeculae.

In these sections you can see the bone tissue as the dark reddish network. This is probably the development of a bone of the cranial roof (in a rodent); you can see a suture between two bones forming. Look at the illustration on the next page and make sure you identify the trabeculae of membrane bone and the osteocytes, which are responsible for secreting the matrix.

On slide I-49 or I-44, the formation of cartilage replacement bone can be seen. This is a section of a developing limb of a rodent, so you can see cartilage, joints and endochondral bone formation. Youll also be able to see some elements of the integument. Take some time and study both the cartilage as well as the bone. Bone formation is taking place in the center of the elements. Notice the difference in texture of the newly formed bone: it is spongy centrally and compact around the shaft of the bone, or bone collar (this is called cortical or perichondral bone). Also, observe the changes in the character of the cartilage as it is being eroded away by the process of ossification.

See the illustration on the below for an overview and photos of the section on the next page.

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On slide I-45 you can see Haversian systems in mature, compact bone. The bone matrix is intact and the section was made by grinding. This section is of compact bone, which has been remodeled. Osteocytes (bone cells) lie in the lacunae and connect via small radiating canaliculi (= little canals). Frequently it will be seen that groups of lacunae are concentrically arranged around open central canals (Haversian canals). This arrangement reflects the concentric deposition of matrix in layers or lamellae around a blood vessel. Review the structure of Haversian canals in your textbook for more information.

Refer to the targeted review on bone and cartilage for more detail.

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Par t III The anatomy of a ver tebra

In preparation for lecture on Thursday we would like you to gain a basic familiarity with a typical vertebral body. Well see in lecture and in lab next week that vertebrae vary along the spinal cord and also are very different in different species, but for now wed like you to become familiar with the terminology below.

The illustrations are of human thoracic vertebrae, youll be given a cat thoracic vertebra. Use your dissection guide for details on cat anatomy. It is difficult to see some features on two dimensional illustrations, so look at the vertebra from several angles. Please make sure you know what we refer to when we talk about:

Vertebral body or centrum Neural arch, Spinous process Transverse process Vertebral (neural) foramen Superior articular process (and facet) =

prezygapophysis Inferior articular process (and facet) =

postzygapophysis Intervertebral foramen

After looking at a single vertebra, fit several together and look at the way that the superior and inferior processes (the terminology used in human anatomy, the comparative terms are pre- and postzygapophyses) fit together to provide torsional stability.

Illustrations from the following website:

http://biology.clc.uc.edu/fankhauser/Labs/Anatomy_&_Physiology/A&P201/Skeletal/selected_bones/Bone_Features.html

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Par t IV Diversity of ver tebrate embryos

As you recall from lecture the life of any individual vertebrate begins with the union of a male and a female gamete; it moves along a continuum of processes that produce an adult and ultimately ends in death. It is important to remember development is a continuum, but it is useful for us to recognize defined stages, like the frames of a motion picture film. As we discussed in lecture, the major developmental stages of the vertebrates are these:

A. Embryonic stages

(1) Zygote: the one cell stage resulting from sperm fertilization of the egg (Gr. zygotes = yoked together, i.e. union of egg and sperm).

(2) Early cleavage: 2 cell, 4 cell, 8 cell, etc. stages; cleavage partitions the egg into many smaller undifferentiated cells called blastomeres.

(3) Blastula: a stage in which the blastomeres generally become rearranged around a fluid-filled cavity. (L. diminutive of Gr. blastos = bud)

(4) Gastrula: the first major rearrangement of cells occurs during gastrulation (Gr. gastrula = little stomach). This process establishes in the embryo the "tube-within-a-tube" configuration around which the mature body is fashioned. The gastrulation process also establishes the three germ layers from which the primordia for all organs and tissues of the adult body will form.

(5) Neurula: in this stage dorsal folds of ectoderm elevate and unite to form the neural tube. The process is called neurulation.

(6) Pharyngula: all or most of the organ systems are established as rudiments, especially visceral arches, in their proper positions at this stage.

(7) Fetal stages (largely in organisms in which no larval stage exists) in which all systems are laid down, but the embryo continues to grow and further differentiate.

At the diversity table we have placed models of these stages from several different types of organisms. There are models of the early stages in amphioxus, representing the primitive condition. Models in frogs will demonstrate cleavage through neurulation in species with large amounts of yolk. Then, we have models of chicken embryos in later stages, so you can see the elements present in a pharyngula stage embryo. Finally, look at the embryos of pigs, which show you a number of fetal stages in a mammalian embryo. Use these to review these stages and understand some of the diversity in early development we see in vertebrate embryos.

For more information

Be sure to look at targeted reviews on Histology and also on Bone and Cartilage. Look at the clinical correlations on vitamin D and also on Osteoporosis. Review the anatomy shown in the histology atlas.

You can further review histology on the following website.

http://www.kumc.edu/instruction/medicine/anatomy/histoweb/index.htm

Remarkable videos of humpback whales feeding. This is notable not only because it shows you how the baleen is used, but also the adaptations for expansion of the throat. Read the caption below the video.

http://www.arkive.org/humpback-whale/megaptera-novaeangliae/video-08b.html

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Key terms:

You should understand the following terms. By understand we mean you should both be able to define and also find the structure (where applicable) on a slide or specimen. Refer to the targeted review for more detail on bone and cartilage.

keratin, epidermis, dermis, bone, cartilage, endochondral bone, membrane bone, Haversian canal, osteocyte, chondrocyte.

vertebral body or centrum, neural arch, spinous process, transverse process, vertebral (neural) foramen, superior articular process (and facet) = prezygapophysis, inferior articular process (and facet) = postzygapophysis, intervertebral foramen

You should also be able to identify all the key structures we point out (such as keratin, glands, chromatophores, fat pads, etc) in the histological slides you have examined.

Key questions:

What kinds of adaptations do we see in the integument of animals for protection against abrasion or water loss?

Why might an animal not want to have an epidermis that has a thick keratin layer?

What characterizes connective tissue and what are the different kinds of connective tissues?

What are the key differences between cartilage and bone structurally and functionally?

What are the origins of cartilage and bone in evolution and in development?

Key competencies:

You should be able to identify major structures of the epithelium of a frog and mammal on a histological slide (or photo of one).

You should understand what elements are derived from dermis and what elements from epidermis.

You should be able to identify cartilage and bone as well as the elements of the Haversian system in a section (or photo of one).

You should know the difference between endochondral and dermal (= membrane) bone.

You should know the major anatomical elements of a typical mammalian vertebra.

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APPENDIX: GUIDELINES FOR USING A COMPOUND MICROSCOPE

We ask that when you use a compound microscope you follow these guidelines carefully. This is important in order to optimize the performance of the microscope (so you can actually see images), and to protect both the slides and the microscope itself. Many of the slides youll look at are quite rare or expensive to replace.

Look at the illustration of the microscope below (yours will be slightly different). The focusing knobs (FK) have both a fine and a course focus. You can use the ring controlling the diaphragm at the base of the condenser to provide a larger circle of light if necessary (CR).

Look at the array of objectives on your microscope the power of the objective is identified on each objective. For most specimens, you should use 10X power, although you may want to start out with X4 to find the specimen. When moving from one objective to another, never use the objectives to rotate the array always move it by grasping the objective ring (OR) and gently rotating.

Before using the microscope make sure you adjust its position. Make sure your chair is at a proper height to comfortably look through the eyepieces. Change the distance between the two eye pieces so that you can look through both comfortably. You may have to adjust the eyepiece focus. Note that one eye piece, generally the one on the left, has a focusing ring. Bring the specimen into focus while looking only through your right eye using the focusing knob. Then open your left eye and use the eye piece focusing ring to bring the left eye into focus.

When changing slides, always move the stage DOWN (using the focusing knobs) and make sure you are using an objective of 10X or less. After putting the slide on the stage, move the stage back into focus. For some of the slides it may be useful to use a low power (4X) to scan the slide for the section, and then move into higher powers for observation.

Always move the stage slightly DOWN before switching to an objective of HIGHER power.

Never touch a lens surface with anything (finger, tissue paper, etc). If it is dirty, call an instructor.

Note: the dissecting microscopes, which are used for observing whole mounts, are much more forgiving of abuse, however, still be careful, and in particular avoid touching lens surfaces.

CR

FK

OR